Advances in Cancer Research Volume 74

Advances in

CANCER RESEARCH Volume 74

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Advances in

CANCER RESEARCH Volume 74

Edited by

George F. Vande Woude ABL-Basic Research Program National Cancer Institute Frederick Cancer Research and Development Center Frederick, Maryland

George Klein Microbiology and Tumor Biology Center Karolinska lnstitutet Stockholm, Sweden

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

This book is printed on acid-free paper.

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Copyright 0 1998 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center. Inc. (222 Rosewood Drive, Danvers, Massachusetts 0 1923). for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1998 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-230>(/98 $25.00

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Contents

Contributors to Volume 74 vii

Key Effectors of Signal Transduction and GI Progression Martine F. Roussel I. Introduction 1 11. The G1 Phase of the Mammalian Cell Cycle 3 111. Signal Transduction and G1 Progression 5 IV. The RAS/ERK Pathway and the Cell Cycle 5 V. V1. VII. VIII. IX.

RAS, D-Type Cyclins, and RB Connections 9 Cycling with MYC 11 Interplay between MYC and Cyclin D1 13 Signaling and Cell Cycle Roles of the SRC Family of Kinases 14 Concluding Remarks 15 References 16

p53 in Tumor Progression: Life, Death, and Everything Michael R. A. Mowat I. Introduction 25 11. Biochemical Activities of p53 26 111. p53 and Cell Cycle Control 27 IV. p53 and Apoptosis 29 V. p53 and Tumor Progression 37 References 42

Signal Transduction through MAP Kinase Cascades Timothy S. Lewis, Paul S. Shapiro, and Natalie G. Ahn I. The MAP Kinase (MAPK) Module 49 50

11. Mammalian MAPK Pathways

V

vi

Contents

Regulation of MAPK Pathways by Protein Phosphatases 75 Cellular Substrates of MAP Kinases 81 Responses to MAPK Pathways: Growth and Differentiation 91 Yeast MAPK Pathways 100 Intracellular Targeting and Spatial Regulation of MAPK Pathway Components 111 VIII. Future Directions 113 References 114 111. IV. V. VI. VII.

FHIT in Human Cancer Gabriella Sozzi, Kay Huebner, and Carlo M. Croce I. Introduction

141 144 146

11. Cloning and Structural Features of the FHIT Gene

111. The Fhit Protein and Its Biochemical Properties IV. FHIT Abnormalities in Human Cancer 147 V. Conclusions and Perspectives 158 References 159

Phosphoinositide 4- and 5-Kinases and the Cellular Roles of Phosphatldylinositol4,5-Bisphosphate 1. Justin Hsuan, Shane Minogue, and Maria dos Santos I. 11. 111. IV. V.

Introduction 167 Receptor-Linked Phosphoinositide Metabolism 174 Phosphoinositides and the Cytoskeleton 187 Vesicle Biogenesis and Trafficking 195 Roles in Cancer, Summary, and Prospects 206 References 208

Index 217

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Natalie G. Ahn, Department of Chemistry and Biochemistry,Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309 (49) Carlo M. Croce, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 19107 (141) Maria dos Santos, Ludwig Institute for Cancer Research, University College London Medical School, London W1P 8BT, United Kingdom (167) J. Justin Hsuan, Ludwig Institute for Cancer Research, University College London Medical School, London W1P SBT, and Department of Biochemistry and Molecular Biology, University College London, London WClE 6BT, United Kingdom (167) Kay Huebner, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 19107 (141) Timothy S. Lewis, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309 (49) Shane Minogue, Ludwig Institute for Cancer Research, University College London Medical School, London W1P SBT, United Kingdom (167) Michael R. A. Mowat, Manitoba Institute of Cell Biology, Winnipeg, Manitoba, Canada R3E OV9 ( 2 5 ) Martine F. Roussel, Department of Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105 ( 1 ) Paul S . Shapiro, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309 (49) Gabriella Sozzi, Division of Experimental Oncology A, Istituto Nazionale Tumori, 20133 Milan, Italy (141)

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Key Effectors of Signal Transduction and GI Progression Martine F. Roussel Department of Tumor Cell Biology St. Jude Children’s Research Hospital Memphis, Tennessee 381 0.5

I. Introduction 11. The G1 Phase of the Mammalian Cell Cycle 111. Signal Transduction and GI Progression IV. The RASERK Pathway and the Cell Cycle

V. VI.

VII. VIII. IX.

A. The RASERK Pathway B. ETS Transcription Factors as RAS Targets and Regulators of Proliferation RAS, D-Type Cyclins, and RB Connections Cycling with MYC A. MYC and Its Partners B. MYC and Its Targets Interplay between MYC and Cyclin D 1 Signaling and Cell Cycle Roles of the SRC Family of Kinases Concluding Remarks References

I. INTRODUCTION Cells enter the cell cycle and commit to DNA synthesis in response to extracellular mitogenic signals. Cytokines and polypeptide growth factors, as well as cell-cell contacts and cell-substratum interactions, provide the positive and negative signals that govern cellular proliferation, differentiation, growth arrest, and apoptosis. Cells respond to these signals during the first gap ( G l ) phase of the cell cycle via their surface receptors, which transmit extracellular cues to the inside of the cell. When ligands bind these receptors, they trigger a cascade of events that includes receptor aggregation and the activation of their tyrosine kinases. The receptors for macrophage colonystimulating factor-1 (M-CSF/CSF-1), platelet-derived growth factor (PDGF), or epidermal growth factor (EGF) all possess intrinsic tyrosine kinase activity that is activated in this fashion (Ullrich and Schlessinger, 1990; Schlessinger and Ullrich, 1992). In contrast, the cytokine family of receptors associate with the Janus tyrosine kinases (JAKs), which autophosphorylate and transphosphorylate the receptor (Ihle, 1996). Specific phosphoryAdvances in CANCER RESEARCH 0065-23OW98 $25.00

Copyright 0 1998 by Academic Press All rights of reproduction in anv form reserved.

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Martine F. Roussel

Fig. 1 The mammalian cell cycle. The cell cycle is composed of four distinct phases: G1, S, G2, and M. G1, the first gap phase of the cell cycle, represents the interval during which cells are sensitive to mitogen stimulation; R, the end point of mitogen sensitivity or restriction point defining the point in G1 beyond which cycling is independent of mitogens; S, the DNA synthetic phase; G2, the second gap phase; M, mitosis; GO, the resting or quiescent phase; RB, retinoblastoma gene product; D1,2,3, D-type cyclins; CDK4,6, cyclin-dependent kinase -4 and -6; P,phosphorylated residues. The dark line represents the interval in G1 during which cells are sensitive to extracellular stimuli.

lated tyrosine residues serve as docking sites for effector proteins containing SRC homology-2 and phosphotyrosine-binding (PTB) domains (Schlessinger, 1994; Harrison, 1996). These associations, in turn, activate a complex signaling network that ultimately instructs the cell cycle machinery. Removal of extracellular stimuli during early or mid-G1 before the cells commit to the DNA synthetic (S) phase of the cell cycle disables the cells from entering the next phase of the cell cycle, the S phase. However, once committed to DNA synthesis, or passed the restriction (R)point, cells no longer require extracellular stimuli to complete the first mitosis (Fig. 1) (Pardee, 1989; Sherr, 1994). Persistent receptor activation is therefore required throughout most of G1 to ensure that genes whose products are rate limiting for G1 progression are transcribed. Oncogenes effectively bypass the regulation exerted by extracellular stimuli, which dysregulates cell growth. Effectors of signal transduction are therefore linked to cell cycle regulators. The pathways that connect these effectors and regulators are now emerging, in part as a result of the vast amount of information gained from yeast genetics (Wittenberg and Reed, 1996). This review highlights some of the advances coupling certain key effectors

Key Effectors of Signal Transduction and GI Progression

3

of cytokine and growth factor receptor signaling to the cell cycle clock, thereby connecting these two broad fields of research. Several of the critical effectors that enforce progression through G1 and influence the commitment of the cell to DNA synthesis will be discussed.

11. THE GI PHASE OF THE MAMMALIAN CELL CYCLE Cells are sensitive to extracellular signals during most of the G1 phase of the cell cycle, and these signals ultimately converge to induce the expression and activity of the D-type cyclins (Sherr, 1993, 1994). The D-type cyclins, of which there are three members, D1, D2, and D3, are exquisitely regulated by extracellular stimuli and their expression requires new protein synthesis (Matsushime et al., 1991; Sherr, 1993).Because D-type cyclins are short-lived proteins (half-lives of 15-20 min), the complexes they form with their catalytic partners, cyclin-dependent kinases (CDK4 and CDK6), are dynamic. D cyclins are rate limiting for G1 progression as enforced expression of cyclin D1 into fibroblasts accelerates their G1 phase; conversely, microinjection of antibodies to cyclin D1 in the same cells induces G1 arrest (Quelle et al., 1993; Resnitzky et al., 1994; Lukas et al., 1995). D-type cyclins, therefore, act as growth factor sensors, enforcing G1 progression into S phase (Sherr, 1994). This pivotal role makes D-type cyclins likely targets of signaling effectors that promote proliferation. Active cyclin D/CDK complexes enforce G1 progression in part by phosphorylating the retinoblastoma gene product, RB (Sherr, 1994; Weinberg, 1995). This phosphorylation eliminates the suppressive function of RB by promoting the release of RB-tethered transcription factors, including the E2Fs (Nevins, 1992; La Thangue, 1994). E2Fs are required for the coordinate regulation of genes essential for DNA synthesis (Hollingworth et al., 1993). The principal role of D-type cyclins in G1 appears to phosphorylate RB (Ewen et al., 1993; Sherr, 1994). In fact, microinjection of D1 antibodies into RB null fibroblasts, or into transformed cells that contain a dysfunctional RB, is unable to prevent S phase entry. The same experiment with RB-positive cells leads to G1 arrest (Quelle et al., 1993; Baldin et al., 1993; Lukas et al., 1995). Cyclin D/CDK complexes are positively regulated by growth factors and by phosphorylation by cyclin-activated kinase (CAK) (Morgan, 1995). However, the activity of these complexes is also negatively regulated by two groups of CDK inhibitors that bind to CDKs and inhibit their kinase activity (Hunter, 1993; Elledge and Harper, 1994; Sherr and Roberts, 1995). The first class of inhibitors includes p21WAF-l,p27K*P1,and each of which can bind and inhibit all of the cyclinlCDK complexes. In contrast, a

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second class of CDK inhibitors, termed INK4s for Dhibitors of c d u , is typified by the tumor suppressor ~ 1 6 (Serrano ” ~ ~et al., ~ 1993, 1996; Xiong et al., 1993) and includes four members: p161NK4a,p151NK4b, and ~ 1 9 ” These ~ ~ ~inhibitors . specifically bind to and inhibit CDK4/6 (Elledge and Harper, 1994; Sherr and Roberts, 1995; Hall and Peters, 1996). The ability of the INK4s to arrest cells in G1 and inhibit CDK4/6 activity is dependent on functional RB (Guan et al., 1994; Lukas et al., 1995a,b). The regulation of these inhibitors is under intense investigation and their induction is thought to occur after cells receive antiproliferative signals or as cells differentiate. For example, ~ 1 5 is” synthesized ~ ~ ~ in response to stimulation by tumor growth factor+ (Hannon and Beach, 1994). However, the exact mechanism of this transcriptional regulation has yet to be clearly defined. In addition, INK4 proteins may have additional functions unrelated to their role as inhibitors of D-CDK4/6 complexes (Hirai and Sherr, 1996; Skapek et al., 1996). The remaining G1 cyclin, cyclin E, is expressed later than D cyclins in G1 and is regulated by E2F (Fig. 2 and 5 ) (Botz et al., 1996). Cyclin E associates with CDK2 (Dulic et al., 1992), and this holoenzyme also phosphorylates RB and other substrates likely to be important for the execution of S phase (Fig. 5) (Ohtsubo and Roberts, 1993; Hatakeyama et al., 1994; Resnitzky and Reed, 1995; Ohtsubo et al., 1995). Like cyclin D, cyclin E is required for S-phase entry, and its expression is limiting for G1 progression (Wimmell et al., 1994).

FOS JUN

I\

E

A

MYC

Go

GI

R

S

~///////~~//////////~

Mitogen Sensitivlty

Fig. 2 Transcription regulation during the cell cycle. The dashed bar represents the period during which cells are sensitive to extracellular stimuli. FOS, JUN, and MYC are immediate early genes transcribed in response to extracellular stimuli. Cyclins D1, E, A, and Bare expressed sequentially as cells pass through the cell cycle. G1, S, G2, and M represent the different phases of the cell cycle. GO represents the resting phase following the withdrawal of cells from the cycle.

Key Effectors of Signal Transduction and GI Progression

5

111. SIGNAL TRANSDUCTION AND GI PROGRESSION Signal transduction is the process by which growth factors and extracelM a r stimuli transmit signals from the outside of the cell to the nucleus. Receptor signals usually persist for the first few hours following growth factor or lymphokine binding, receptor aggregation, and internalization of the receptor-ligand complex (Heldin, 1995). Because many of the signaling pathways induced by ligand binding to receptors have been covered in other reviews, only the pathways and effectors relevant for this review will be discussed. The tyrosine kinase growth factor and cytokine family of receptors activate multiple pathways. These include the RAYMAP kinase (RAY ERK) pathway that leads to the expression of the immediate early genes FOS and JUN and the pathways that lead to MYC transcription, which may involve SRC kinase activation (Fig. 3) (Schlessinger and Ullrich, 1992; Schlessinger, 1993). The cytokine family of receptors also activates the JAWSTAT pathway (Darnel1 et d., 1994; Ihle, 1996). The role of these signaling pathways in cell proliferation has started to be elucidated. Pathways leading to FOS and JUN expression were shown to be insufficient to stimulate G1 phase progression. CSF-1 or PDGF-regulated expression of FOS and JUN is insufficient for G1 phase progression (Roussel et al., 1990; Barone and Courtneidge, 1995). Moreover, cytokine receptor activation of RAS, and subsequently of AP-1 and STATs, is dispensable for entry into S phase (Taniguchi and Minami, 1993; Ihle, 1996). In contrast, JAK activation is essential for MYC induction via the “X” pathway (Fig. 3) (Ihle, 1996),which infers that the upstream regulators of MYC in response to cytokine activation are still unknown. In addition, a nonreceptor tyrosine kinase, BCR-ABL, also mediates the transformation of B cells and fibroblasts via both RAS and “MYC” pathways. The constitutively activated kinase V-ABLtransforms B cells only during the early G1 phase of the cell cycle. This suggests that the pathways required for transformation by the ABL kinase are also required for G1 progression (Chen and Rosenberg, 1992); indeed, MYC expression is essential for this transformation process (Sawyers et d.,1992).

1V. THE RASERK PATHWAY AND THE CELL CYCLE A. The RASERK Pathway RAS, which was first identified as an oncogene (Bishop, 1982; Weinberg, 1989), plays an essential role in the proliferation of most cell types as a mediator of ligand-induced receptor signaling (Figs. 3 and 4). Dominant-nega-

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Fig. 3 Signal transduction pathways leading to activation of the immediate early response genes. In response to extracellular stimuli, activated receptors induce several pathways: 1RAS/MAP kinase pathway; 2-SHC, activated RAS/MAPK pathway; 3-JAWSTAT and “X” pathway; 4-SRC/MYC pathway. In the nucleus, the three genes that represent the “immediate early response” are FOS, JUN, and MYC.

tive forms of RAS block G1 progression mediated by receptors for several growth factors, including PDGF, EGF, and CSF-1 (Stacey et al., 1991), and transformation by the cytoplasmic tyrosine kinase BCR-ABL (Sawyers et al., 1995). Similarly, microinjection of anti-RAS antibodies reverses cell transformation by oncogenic forms of growth factor receptors, such as CSF-1R (v-fms),or by the cytoplasmic oncogenic effectors, V-RAS or V-SRC. Therefore, RAS is essential for transformation by most oncogenes (Mulcahy et al., 1985). Conversely, microinjection of activated RAS, Ha-RAS, in NIH3T3 cells leads to transformation (Stacey and Kung, 1984). The signaling pathway initiated by the activation of these small GTP proteins is highly conserved throughout evolution (Schlessinger and Bar-Sagi, 1994). Predictably, these proteins are fundamental to the development of all animal species (Dickson and Hafen, 1994)and control a number of cytoplasmic events that ultimately regulate gene expression (Fig. 4)(Egan and Weinberg, 1993; Segar and Krebs, 1995).

7

Key Effectors of Signal Transduction and G I Progression Growth Factors Cytokines

1

1

RAS

-

FOS, JUN

,-

DllK4 E2F Responsive Genes

Fig. 4 RAS, D-type cyclin, RB, and G1 progression. Summary of the pathways connecting activated receptors to RAS, cyclin D1, and RB. Arrows (-) represent established connections between effectors, whereas dashed arrows (---) represent potential links between FOS, JUN, and cyclin D1. D1, cyclin D1; D l K 4 , holoenzyme complex between cyclin D1 and cyclin-depenmhibitor of C d u ; RB, retinoblastoma gene product; EZFiDPdent kinase CDK4; p l 61NK4a, 1, transcription complex between E2F and DP-1; P, phosphorylated residue.

Microinjection of activated Ha-RAS into quiescent cells can induce DNA synthesis (Stacey and Kung, 1984). Conversely, microinjection of antibodies to RAS into serum-deprived Balb3T3 cells prevents DNA synthesis, demonstrating that RAS is required for S-phase entry (Dobrowolski et al., 1994). Constitutively, activated RAS and RAF can drive the cells through S phase, suggesting a direct link between RAS and the GUS transition (Dobrowolski et al., 1994; Samuel et al., 1993; Marshall, 1995). Similarly, activated MAP kinases (ERK-1 and ERK-2) are required for fibroblast transformation (Pages et al., 1993), and constitutive activation of MEKs is necessary and sufficient for fibroblast transformation (Cowley et al., 1994; Mansour et al., 1994). Interestingly, constitutive activation of each one of the key members of the RAS/ERK pathway leads to the GUS transition, suggesting that this pathway represents a critical link between receptor signaling and G1 progression. RAS regulates a number of downstream effectors (Fig. 3) (Marshall, 1996), including the cytoplasmic serine threonine kinase RAF-1 (McCormick, 1994), the dual specificity mitogen-activated protein kinase kinase (MAPKK or MEK-1 and MEK-2) (Cobb and Goldsmith, 1995), and the ex-

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Martine F. Roussel

tracellular regulated kinases (ERK-1 and ERK-2 or MAPK) (Egan and Weinberg, 1993; Su and Karin, 1996; Davis, 1996).RAF-1 activates MEK-1 (Kyriakis et al., 1992), as well as ERK-1 and ERK-2 (Samuel et al., 1993). In turn, ERKs phosphorylate and modulate the activity of nuclear factors that regulate the transcription of genes required for proliferation and differentiation (Treisman, 1996). In particular, the RAS pathway regulates the transcription of FOS and JUN (Blenis, 1996; Treisman, 1996), which, as dimers, define the AP-1 nuclear transcription factor (Karin, 1995; Su and Karin, 1996).

B. ETS Transcription Factors as RAS Targets

and Regulators of Proliferation Analysis of the FOS promoter has provided the basis for a model in which a complex coined the tertiary complex factor (TCF)contains an ETS domain. TCFs are direct targets of ERKs from the RASERK signaling pathway and are essential for the transient and immediate early transcription of FOS (Marais et al., 1993; Hill and Treisman, 1995; Janknecht et al., 199s). The ETS family of winged-helix-loop-helix transcription factors represents an extended family of genes, some of which were isolated as oncogenes, e.g., ETS-1 and FLI-1 (Macleod et al., 1992; Seth et al., 1989; Klemsz et al., 1993). ETS factors are characterized by a DNA-binding domain, the ETS domain, which recognizes a GGA core sequence surrounded by purine-rich motifs (Wasylyk et al., 1992, 1993; Wang et al., 1992). They often bind cooperatively to promoters with other transcription factors such as AP-1 (Bassuk and Leiden, 1995; Wasylyk et al., 1990) or SP1 (Gegonne et al., 1993) to regulate gene transcription (Janknecht and Nordheim, 1993). In mammalian cells, ectopic expression of a dominant-negative form of ETS inhibits the transformed phenotype imposed by activated RAS (V-RAS) and activated CSF-1R (V-FMS)and blocks CSF-1-dependent proliferation in NIH3T3 fibroblasts expressing human CSF-1R (Langer et al., 1992). Interestingly, this G1 block is associated with a reduction in CSF-l-induced MYC levels. Enforced MYC expression in these cells restores CSF-l-dependent proliferation, suggesting that the RAS pathway, and ETS in particular, regulates the MYC response in this system (Langer et al., 1992; Roussel et al., 1994). An additional connection between ETS and G1 progression has come from studies of the ETS-related transcription factor ELF-1 (Leiden et al., 1992; Davis and Roussel, 1996). ELF-1 activity is regulated, in part, by binding to RB through a pentapeptide (LXCXE) RB-binding domain (Wang et al., 1993),a motif shared by D- and E-type cyclins and by the DNA tumor virus oncoproteins SV40 T antigen, adenovirus ElA, and HPV E7 (Ewen et al., 1993; Ewen, 1994; Weinberg, 1995). This interaction therefore offers a di-

Key Effectors of Signal Transduction and GI Progression

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rect physical link between transcription regulation and the cell cycle, although it is not entirely clear how ELF-l regulates cell growth. The cumulation of these data, combined with the fact that some ETS factors are involved in the etiology of tumors (Zucman et al., 1992, 1993) underscores the role of this family of transcription factors in the regulation of G1 progression, proliferation, and transformation.

V. RAS, D-TYPE CYCLINS, A N D RB CONNECTIONS Cyclin D1 expression is upregulated by both activated RAS (v-Ha-RAS) and ERK-1 and ERK-2 (Lavoie et al., 1996). These data predict that the cyclin D1 promoter is regulated by elements responsive to RAS signals. The D1 promoter contains an AP-1-binding site and a cyclin AMP responsive element (CRE) that are inducible by small t antigen and c-JUN (Watanabe et al., 1996; Herber et al., 1994). Moreover, JUN nullizygous fibroblasts are resistant to RAS-induced transformation (Johnson et al., 1996). Therefore, a likely scenario is that the RASIMAP kinase pathway is linked to cyclin D expression as follows: activated RAS activates ERK kinases, which induce FOS and JUN, which in turn induce cyclin D1 transcription (Fig. 4). These findings suggest that cyclin D1 is in the RAS pathway and would cooperate with MYC, but not RAS, in transformation assays. Paradoxically, Harvey (Ha)-RAS cooperates with cyclin D1 to transform primary rat embryo fibroblasts (REF) (Lovec et al., 1994), although this appears to be cell specific as no synergy is observed if the experiment is done in rat kidney cells (BRK) (Hinds et al., 1994).In agreement with the fact that RAS and D1 are in the same pathway, the inducible expression of activated RAS in epithelial or fibroblast cells induces cyclin D1 expression (Winston et al., 1996) and cyclin D1 levels are increased in Ha-RAS-transformed cells, which accounts for their shortened G1 phase (Liu et al., 1995). RAS-induced transformation of some cells also requires cyclin D/CDK4 activity, and anti-sense cyclin D 1 oligonucleotides reduce the rate at which RAS-transformed cells proliferate (Filmus et al., 1994). Enforced expression of p161NK4"blocks the ability of activated RAS to transform fibroblasts cells (Serrano et al., 1995). However, at least in fibroblast cells, cyclin D1 expression alone is insufficient to drive DNA replication, suggesting that the activation of the DICDK holoenzymes requires mitogenic signals in addition to those involving RAS for Sphase entry (Winston et UE., 1996). This is a common theme as both cyclin D and CDK4 require mitogenic signals to form complexes (Matsushime et al., 1994). Several experiments suggest that D1 is in the RAS pathway. Ectopic expression of a dominant-negative form of RAS (Asnl7) in cells that lack func-

Martine F. Roussel

10 Growth Factors Cytokines

-m IS?C

““p/

rc

---)

”.‘.

FOS, JUN

a

Other Targets CDC25A, 8

D1

I

8 8

Fig. 5 MYC, D1, E2Fs and G1 progression. Summary of signaling pathways linking the major effectors of receptor signaling to those of G1 progression and S-phase entry. Arrows (-+) represent established pathways; whereas dashed arrows (-- +) are potential links between effectors. JAK, Janus kinase; CDC25A, a cell cycle tyrosine phosphatase (25A); D1, cyclin D1; DlK4, complex between cyclin D1 and the cyclin-dependent kinase CDK4; RB, retinoblastoma gene product; S, DNA synthetic phase; P, phosphorylated residue; E, cyclin E; A, cyclin A; DHFR, dihydrofolate reductase; pol 11, DNA polymerase 11; EK2, inactive kinase complex between cyclin E and the cyclin-dependent kinase CDK2; E/K2,* activated kinase complex that phosphorylates cyclin E and RB.

tional RB is unable to prevent S-phase entry, whereas Am17 expression in cells containing wild-type RB blocks both D1 expression and S-phase entry (Peeper et al., 1997). Therefore, RAS is linked to RB in the transition from G1 into S-phase v i s - h i s cyclin D1. Phosphorylation of RB releases its association with the E2F family of transcription factors (Fig. 5). E2F associates with a dimerization partner termed DP-1 and together form a potent transcriptional activator of many genes essential for G1 progression and DNA synthesis (La Thangue, 1994), including cyclin E (Botz et al., 1996), cyclin A (Schulze et al., 1995),cdc2 (Dalton, 1992), dihydrofolate reductase (DHFR) (Blake and Azizkhan, 1989), MYC (Thalmeier et al., 1989; Hiebert et al., 1989), and DNA polymerase a (Pearson et al., 1991).Predictably, enforced E2F expression is sufficient to replace some mitogenic signals, and microinjection of E2F-1 overrides the require-

Key Effectors of Signal Transduction and GI Progression

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ment for cellular RAS in initiating DNA synthesis (Stacey et al., 1994). Thus E2F-1 can substitute for the earlier signaling requirements to progress through G1 and enter S phase.

VI. CYCLING WITH MYC A. MYC and Its Partners MYC was first isolated as a transforming gene from avian acute leukemia viruses, MC-29 and MH-2 (Alitalo et al., 1987), and its cellular counterpart was identified and cloned soon thereafter (Roussel et al., 1979; Bishop, 1982).MYC was the first oncogene found to be overexpressed as a result of a chromosomal translocation (Bishop, 1983).Since this seminal observation, amplification of MYC family members (MYC, N-MYC, and L-MYC) has been shown in many human tumors and is known to deregulate cell growth by promoting continuous, mitogen-independent, cell cycle progression (Eilers et al., 1991; Askew et al., 1991; Evan et al., 1992; Henriksson and Liischer, 1996; Lemaitre et al., 1996). The importance of MYC to cell cycle progression has also been established through studies of its function as a transcription factor (Adkins et al., 1984; Blackwood et al., 1991; Amati and Land, 1984).In concert with its partner MAX (Blackwood and Eisenman, 1991),MYC positively regulates the transcription of genes important in cell growth and homeostasis (Packham and Cleveland, 1995).MAX also forms heterodimers with other members of the HLWbZIP transcription factor family, including MAD1 , MXIl (MAD2), MAD3, and MAD4 (Ayer et al., 1993). MAD/MAX heterodimers actively repress gene transcription and, in so doing, antagonize MYC function (Zervos et al., 1993; Hurlin et al., 1995). Transcriptional repression by MAD/ MAX occurs through the formation of ternary complexes with a conserved, generalized transcriptional repressor (SIN3), which was initially identified in yeast (Schreiber-Aguset al., 1995; Ayer et al., 1995). Enforced expression of MAD inhibits tumor growth (Chen et al., 1995) and induces G1 arrest in serum- or CSF-1-stimulated fibroblasts. These biologic effects require the association of MAD with both MAX and SIN3 (Ayer et al., 1995; Roussel et al., 1996). MYC is normally expressed at a low but constant level in proliferating cells. However, when quiescent cells are stimulated by growth factors or serum, they transiently express much higher levels of MYC (Fig. 2). MYC is downregulated as cells differentiate or in the absence of mitogenic stimuli, whereas MAD genes are induced (Ayer and Eisenman, 1993) and (John Cleveland, personal communication).

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Martine E Roussel

MYC function is necessary for S phase entry as ectopic expression of a dominant-negative form of MYC (Stone et al., 1987; Roussel et al., 1995), MAD (Ayer et al., 1993; Roussel et al., 1996), MAX (Gu et al., 1993), or treatment with anti-sense oligonucleotides (Heikkila et al., 1987; Prochownik et al., 1988) prevents growth factor-induced S-phase entry and leads to G1 arrest. MYC is also limiting for S-phase because its enforced expression in fibroblasts shortens G1 and accelerates cell growth (Keath et al., 1984; Eilers et al., 1989; Roussel et al., 1991; Evan et al., 1984). A mutant form of the CSF-1 receptor that is unable to signal G1 progression (CSF-1R [Y809F]) mediates the normal kinetics of FOS and JUN transcription but fails to induce sustained levels of MYC (Roussel et al., 1990). Similarly, mutants of the p chain of the IL-2 receptor cannot mediate proliferative signals or induce MYC expression, but they do promote normal levels of FOS and JUN (Taniguchi and Minami, 1993). Finally, the nonreceptor tyrosine kinase BCR-ABL mediates transformation via independently activated pathways, including a RAS pathway and a MYC pathway (Sawyers et al., 1992; Goga et al., 1995). CSF-1R and BCR-ABL are tyrosine kinases that mediate mitogenesis and transformation, respectively, by independent and common pathways that involve MYC (Lug0 and Witte, 1989) and cyclin D1 (Afar et al., 1995). Experiments designed to complement either the CSF-1R [Y809F] mutant or a BCR-ABL [SH2] mutant defective in the transformation of B cells or fibroblasts showed that enforced expression of either MYC or cyclin D1 complements both of these defects (Afar et al., 1994,1995). In each system, ectopic MYC expression in the mutants restores receptor-induced proliferation or kinase-induced transformation, demonstrating that the MYC pathway is essential for G1 progression and S-phase entry (Adkins et al., 1984; Roussel et al., 1991; Sawyers et al., 1992).

B. MYC and Its Targets The identification and characterization of the transcriptional targets of MYC have proven to be a difficult task (Fig. 5). Only a relative small number of MYC targets have been identified to date. The following MYC targets have been identified: a-prothymosin (Eilers et al., 1991), ornithine decarboxylase (ODC) (Bello-Fernandez et al., 1993), CAD (Miltenberger et al., 1995), an evolutionary conserved RNA helicase of the DEAD box family, MrDb (Grandori et al., 1996), eIF-4E, a eucaryotic initiation factor of protein synthesis (Jones et d., 1996; Rosenwald et d., 1993), cyclins E and A (Jansen-Durr et al., 19,93), and the tyrosine phosphatases CDC25A and CDC25B (Galaktionov et al., 1996) (Fig. 5). Each of these targets may play a unique role in MYC-mediated S-phase entry, or their combined effects

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might be required to move the cell from G1 into S phase (Packham et al., 1996; Zornig and Evan, 1996). ODC and CDC25A are direct MYC targets. Their promoters contain consensus E box MYC-binding sites whose disruption abrogates the induction of these genes by growth factors (John Cleveland, personal communication). ODC may be directly required for DNA replication because inhibitors of ODC arrest cells in G1 (Packham et al., 1996). The CDC25 phosphatases could activate cyclins E and A expression by regulating the activities of cyclin-CDK complexes. Presumably, this regulation would involve the dephosphorylation of the inhibitory tyrosine phosphate on CDK2 (JansenDurr et al., 1993; Galaktionov et a/., 1996). MYC can alter the expression of several genes, including cyclins E and A (Jansen-Durr et al., 1993; Solomon et al., 1995), although this regulation may be an indirect result of the positive effects of MYC on the cell cycle. MYC also induces the expression of the initiation factor eIF-4E. Phosphorylation of eIF-4E depends on RAS signaling (Frederickson et al., 1992) whereas cyclin D1 levels increase in response to elevated levels of eIF-4E (Rosenwald et al., 1993). Therefore, these are clear links between the regulation of protein synthesis and the cell cycle. Unlike enforced expression of MYC, enforced expression of ODC (Packham et al., 1996) is not sufficient to promote proliferation or to complement a CSF-1R mutant defective in promoting G1 progression ( M.F.R. and John Cleveland, unpublished data). However, CDC25A and CDC25B (but not CDC25C), like MYC, can function as oncoproteins by collaborating with RAS to transform primary mouse embryo fibroblasts (Galaktionov et al., 1995). This suggests that MYC, cyclin D1, and CDC25A may have overlapping functions, at least in this biological setting.

VII. INTERPLAY BETWEEN MYC AND CYCLIN D l Evidence from in vitro and in vivo studies has led to the belief that MYC and D1 act in parallel pathways but cooperate functionally and synergistically to enforce G1 progression. In experiments with transgenic mice, crossing Ep-MYC and E p D 1 mice significantly accelerates the onset of lymphomas (Bodrug et al., 1994; Lovec et al., 1994). Interestingly and surprisingly, the Ep-MYC-induced B-cell tumors express cyclin D1, even though D1 is not normally expressed in B cells (Bodrug et al., 1994). Because cyclin D1 is overexpressed in Ep-MYC-induced tumors only and not in other B cells of Ep-MYC transgenic mice, it suggests that MY C overexpression is necessary but not sufficient to induce cyclin D1 expression. In addition, MYC or D1 is functionally interdependent and each requires the function of the other to

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restore CSF-1-dependent proliferation of fibroblasts engineered to express CSF-lR[Y809F]. For example, microinjection of anti-Dl antibodies into MY C overexpressing cells, or overexpression of a dominant-negative MYC mutant (In373) or MAD proteins in cells expressing CSF-lR[Y809F] and complemented by enforced cyclin D 1 expression, prevents S-phase entry (Roussel et al., 1995, 1996). The interplay between MYC and D1 could be explained by the fact that D/CDK4 complexes phosphorylate RB, thereby releasing E2F/DP-1 complexes that induce MYC transcription (Hiebert et al., 1989; Oswald et al., 1994).Therefore, in cells unable to express MYC, enforced expression of D1 should reinduce MYC expression, albeit with different kinetics from those observed in response to authentic mitogenic stimuli (Roussel et al., 1995). MYC and cyclin D1 seem to enjoy a special relationship that apparently is not shared by D2- and D3-type cyclins. Analysis of the promoter regions and expression patterns of the D-type cyclins (Ewen et al., 1993; Brooks et al., 1996; Wang et al., 1996), coupled with studies of mice in which the D1 or D2 genes are disrupted (Sicinskiet al., 1995,1996), suggests that each D-type cyclin may play specific and overlapping roles in regulating cell proliferation.

W11. SIGNALING AND CELL CYCLE ROLES OF THE SRC FAMILY OF KINASES The SRC family of protein tyrosine kinases, whose founder member SRC was the first oncogene described (Stehelin et al., 1976; Bishop, 1983), has been implicated in the mitogenic response to many growth factors and lymphokines. All SRC kinases contain a conserved SRC homology SH3 domain, a SRC homology SH2 domain, a catalytic domain, and C-terminal regulatory sequences (Superti-Furga and Courtneidge, 1995; Brown and Cooper, 1996). Three members of the SRC kinase family, SRC, FYN, and YES, are closely related and ubiquitously expressed. They mediate signals by physically associating with activated growth factor receptors via their SH2 and their SH3 domains (Erpel et al., 1996). This association is required to activate the activity of the SRC kinases and is dependent on specific tyrosine residues located near the transmembrane-spanning region of such receptors (Mori et al., 1993; Courtneidge et al., 1993; Alonso et al., 1995). A role for the SRC kinases in mitogenic signaling has been established. Microinjection of an anti-SRC antibody that recognizes all three members (anticstl) inhibits EGF-, PDGF-, and CSF-1-induced S-phase entry in fibroblasts expressing equivalent numbers of PDGF-R and ectopically expressed human

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CSF-1R (Twamley-Stein et al., 1993; Roche et al., 1995). However, microinjection of this antibody into cells already committed to S phase (i.e., at least 10 hr after ligand stimulation) has no effect, suggesting that SRC kinases are required to drive cells, at least up to the restriction point, R (Roche et al., 1995).Interestingly, at least in fibroblasts, SRC is also activated in G2, which suggest a requirement for SRC in mitosis (Roche et al., 1995). Microinjection studies have also provided researchers with sufficient data to propose a novel signaling pathway for SRC kinases (Barone and Courtneidge, 1995). Specifically, antibodies to SRC or dominant-negative forms of SRC or FYN prevent MYC mRNA induction and S-phase entry in response to PDGF stimulation. Enforced expression of MYC, but not of FOS or JUN, overcomes this block and restores S-phase entry, thus providing compelling evidence that SRC couples activated growth factor receptors to MYC via a pathway that is independent of RAS. Even though the “SRC/MYC” and/or “X” pathways that lead to MYC expression are dissociated from the RAS signaling pathway, both pathways seem necessary for G1 progression. The situation is further complicated by the fact that these signaling pathways are apparently cell context specific and receptor specific. For example, the IL-2 cytokine receptor activates the SRC kinases LCK, FYN and LYN, which induce pathways that are independent of MY C transcription and insufficient for IL-2-dependent proliferation (Kobayashi et al., 1993; Ihle, 1996). Similarly, BCR-ABL activates the SRC family kinases independently of MYC transcription (Danhauser-Riedl et al., 1996). Nevertheless, the findings of Barone and Courtneidge (1995)provide a link between the SRC kinases and the MYC protooncogene (Eisenman and Cooper, 1995).

IX. CONCLUDING REMARKS Receptor mutants that fail to connect mitogenic signals to cell cycle progression and the enforced expression in cells of key effectors of G1 progression have been useful in identifying many signal transduction pathways. The realization that mammalian D-type cyclins are growth factor sensors has fueled interest in coupling receptor signals to the key effectors that regulate the cell cycle clock. The complexity of these pathways is just beginning to emerge, and novel pathways and effectors likely remain to be identified. Despite the multiplicity of ligands, receptors, and their “wiring” to effectors, most of the identified pathways converge on a few key genes required for G1 progression and S-phase commitment. Strikingly, most of these key players, including RAS, MYC, D-type cyclins, and RB, are frequently targeted in human tumors (Sherr, 1996). One of the major challenges will be to

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understand how these crucial G1 effectors are interconnected. The next few years should yield exciting new discoveries as investigators unravel these pathways.

ACKNOWLEDGMENTS Given the recent explosion of data from the fields of signal transduction and cell cycle, I cannot claim that this review is exhaustive or encyclopedic. Moreover, I must apologize to my colleagues whose work is not included or indirectly cited. I thank Drs. Daniel E. H. Afar, Mangeng Cheng, John L. Cleveland, Jonathan Cooper, Veronika Sexl, and Frederique Zindy for critically reviewing this manuscript and to Dr. Sue Vallance for editorial assistance. My thanks also go to my husband, Dr. Charles J. Sherr, for his continued support, understanding, and encouragement and to the members of my laboratory for their continued efforts and support. MFR is supported by NIH Grants R01-CA-56819, Pol-CA-76907, Cancer Center CORE Grant CA21665 from the National Cancer Institute, and by funds from the American Lebanese Syrian Associated Charities (ALSAC)of St. Jude Children's Research Hospital.

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cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc. Mol. Cell. Biol. 16,4754-4764. Karin, M. (1995).The regulation of AP-1 activity by mitogen-activated protein kinase. /. Biol. Chem. 270,16843-16846. Keath, E. J., Caimi, P. G., and Cole, M. D. (1984). Fibroblast lines expressing activated c-myc oncogenes are tumorigenic in nude mice and syngenic animals. Cell 39, 339-348. Klemsz, M. J., Maki, R. A,, Papayannopoulou, T., Moore, J., and Hromas, R. (1993). Characterization of the ets oncogene family member, fli-1. J. Biol. Chem. 268,5769-5773. Kobayashi, N., Kono, T., Hatakeyama, M., Minami, Y., Miyazaki, T., Perlmutter, R. M., and Taniguchi, T. (1 993). Functional coupling of the src family protein tyrosine kinases ~ 5 9 ~ y " and p53/56'P with the interleukin 2 receptor: Implications for redundancy and pleiotropism in cytokine signal transduction. Proc. Nutl. Acud. Sci. USA 90,4201-4205. Kyriakis, J. M., App, H., Zhang, X.-F., Banerjee, P., Brautigan, D. L., Rapp, U.R., and Avruch, J. (1992).Raf-1 activates MAP kinase-kinase. Nature 358,421427. La Thangue, N. B. (1994). DRTFlE2F: An expanding family of heterodimeric transcription factors implicated in cell cycle control. Trends Biochem. Sci. 19, 108-114. Langer, S . J., Bortner, D. M., Roussel, M. F., Sherr, C. J., and Ostrowski, M. C. (1992). Mitogenic signaling by colony-stimulating factor 1 and rus is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression. Mol. Cell. Biol. 12,5355-5362. Lavoie, J. N., L'Allemain, G., Brunet, A., Muller, R., and Pouysst?gur,J. (1996). Cyclin D1 expression is regulated positively by the p42/p44MAPKand negatively by the p38/HOGMAPX pathway. J. Biol. Chem. 271,20608-20616. Leiden, J. M., Wang, C., Petryniak, B., Markovitz, D. M., Nabel, G. J., and Thompson, C. B. (1992).A novel ets-related transcription factor, elf-1, binds to human immunodeficiency virus type 2 regulatory elements that are required for inducible trans activation in T cells. J. Virol. 66,5890-5897. Lemaitre, J.-M., Buckle, R. S., and Mkhali, M. (1996). c-Myc in the control of cell proliferation and embryonic development. Adv Cancer Res. 70,96-144. Liu, J.-J., Chao, J.-R., Jiang, M.-C., Ng, S.-Y., Yen, J. J.-Y., and Yang-Yen, H.-F. (1995). Ras transformation results in an elevated level of cyclin D1 and acceleration of GI progression in NIH 3T3 cells. Mol. Cell. Biol. 15, 3654-3663. Lovec, H., Grzeschiczek, A., Kowalski, M. B., and Moroy, T. (1994). Cyclin Dl/bcl-1 cooperates with myc genes in the generation of B-cell lymphoma in transgenic mice. E M B O /. 13, 3487-3495. Lovec, H., Sewing, A., Lucibello, F. C., Muller, R., and Moroy, T. (1994). Oncogene activity of cyclin D1 revealed through cooperation with Ha-rus: Link between cell cycle control and malignant transformation. Oncogene 9, 323-326. Lugo, T. G., and Witte, 0. N. (1989). The BCR-ABL oncogene transforms rat-1 cells and cooperates with v-myc. Mol. Cell. Biol. 9, 1263-1270. Lukas, J., Bartkova, J., Rohde, M., Strauss, M., and Bartek, J. (1995a).Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells, independent of CDK4 activity. Mol. Cell. Biol. 15,2600-2611. Lukas, J,, Parry, D., Aagaard, L., Mann, D. J., Bartkova, J., Strauss, M., Peters, G., and Bartek, J. (199%). Rb-dependent cell cycle inhibition by p16CDKN2tumour suppressor. Nature 375, 503-506. Macleod, K., Leprince, D., and Stehelin, D. (1992).The ets gene family. Trends Biochem. Sci. 17,251-256. Mansour, S . J., Matten, W. T., Hermann, A. S., Candia, J. M., Rong, S., Fukusawa, K., Vande Woude, G. F., and Ahn, N. G. (1994). Transformatiion of mammalian cells by constitutively active MAP kinase kinase. Science 265,966-969. Marais, R., Wynne, J., and Treisman, R. (1993). The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell 73,381-393.

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p53 in Tumor Progression: Life, Death, and Everything Michael R. A. Mowat Manitoba Insrirure of Cell Biology, Winnipeg, Manitoba, Canada R 3 E OV9

I. Introduction 11. Biochemical Activities of p53 111. p53 and Cell Cycle Control A. Control of GUS by p53 B. Control of G2/M by p53 C. Nontranscriptional Controls of Cell Cycle by p53 1V. p53 and Apoptosis A. p53 Control of Apoptosis in Tumors B. p53 Transactivation and Apoptosis C. Nontransactivator Function in Apoptosis by p53 V. p53 and Tumor Progression A. Apoptosis and Tumor Progression B. Growth Control and Tumor Progression C. Genornic Instability and Tumor Progression References

I. INTRODUCTION The p53 tumor suppressor protein was first described in 1979 as a protein that binds SV40 virus large T antigen (Linzer and Levine, 1979; Lane and Crawford, 1979) and independently as a tumor antigen (DeLeo et al., 1979). Although the basic outline of p53 protein function in cells has been deciphered, this molecule continues to surprise and baffle scientists. p53 activity has been linked to tumor suppression, cell cycle control, DNA repair, stress responses, cell senescence, genomic stability, and apoptotic cell death. The frequent mutation of the p53 gene in human tumors stresses the importance of trying to understand the function of this protein. Because of the vastness of the p53 literature, this review will concentrate on the role of p53 in tumor progression. In particular, the mechanisms of p53’s control of the cell cycle, genomic stability and apoptosis will be reviewed and how loss of these functions play a role in tumor cell progression. Advances in CANCER RESEARCH 0065-23OW98 $25.00

Copyright 0 1998 by Academic Press All rights of reproduction in any form reserved.

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11. BIOCHEMICAL ACTIVITIES OF p53 p53 primarily functions as a transcriptional transactivator protein with sequence-specific DNA-binding activity (Fields and Jang, 1990; Raycroft et al., 1990; Funk et al., 1992; el Deiry et al., 1992). The transcription transactivating domain of p53, at the amino terminus, interacts with various members of the general transcription initiation complex. These include members of the TFIID complex, such as TATA-binding protein (TBP) (Horikoshi et al., 1995) and TPB associated proteins, TAF,,40 and TAFI160 (Thut et al., 1995; Lu and Levine, 1995). Also, p53 interacts with the TFIIH transcription complex (Xiao et al., 1994; Wang et al., 1995a). The central domain of p53, where most tumor-derived mutations occur, functions in sequence-specific DNA binding (Vogelstein and Kinder, 1992; Cho et al., 1994).The carboxyl terminus of p53 is the location of the oligomerization domain (Shaulian et al., 1992) and the structure of this region has been elucidated (Clore et al., 1994; Lee et al., 1994; Jeffrey et al., 1995). The C-terminal region of p53 can also bind nonspecifically to DNA ends or mismatched DNA and promote annealing of DNA single strands (Lee et al., 1995; Bakalkin et al., 1994; Jayaraman and Prives, 1995; Bakalkin et al., 1995).This region of p53 also functions to control the DNA-binding activity of p53 and can be regulated by phosphorylation (Hupp et al., 1992; Wang and Prives, 1995). The functional domains of p53 referred to in this review are summarized in Fig. 1. A 3' to 5' exonuclease activity intrinsic to p53 protein has been described (Mummenbrauer et al., 1996). Also, p53 binds to and inhibits the DNA re-

53BPl 53BP2 transactivation

TFllH XP-B XP-D

sequence specific DNA binding

N

C

TFllD TBP TAF40 TAFBO TFllH ~ 6 2

50

I

100

I

150

I

200

I

250

I

I

I

u

300

350

393

non-specific DNA binding

Fig. 1 Functional domains of p53 protein. The numbered hatched boxes represent the conserved domains. pS3-binding proteins discussed in the text and their approximate binding regions are shown in bold.

p53 in Tumor Progression

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pair protein Rad51 (Sturzbecher et al., 1996). For more details, the biochemistry of p53 has been reviewed and the reader is referred to KO and Prives (1996).

111. p53 AND CELL CYCLE CONTROL

A. Control of GI/S

by p53

DNA damage and other stresses will stabilize p53 protein, resulting in growth arrest (Kastan et al., 1992; Lu and Lane, 1993). It is now generally accepted that a major control by p53 on the cell cycle at GUS is through transcriptional control of the p21wAF11C'P1gene (see Morgan and Kastan, 1997). The p21 (WAF1, CZP1, SDI)gene was discovered about the same time by several groups as a p53-inducible gene (el Deiry et al., 1993), a cyclin-dependent kinase inhibitor (Harper et al., 1993; Xiong et al., 1993; Gu et al., 1993), and a gene expressed at high levels in senescent cells (Noda et al., 1994). p21wAF11C1P1is an effective inhibitor of Gl/S cyclin-dependent kinases (cdk), Cdk2, Cdk3, Cdk4, and Cdk6 kinases, but a weaker inhibitor of G2 active cdc2 kinase (cdkl) (Harper et al., 1995). Studies performed on p21WAF1/C1P1-deficient mice showed a partial defect in G1 arrest after DNA damage or nucleotide pool perturbations compared with the defect in p53deficient mouse cells (Deng et al., 1995; Brugarolas et al., 1995), suggesting that other gene targets or functions of p53 were also needed for growth arrest. p21WAF11CIP1-deficient mice did not show defects in apoptosis induction or spindle checkpoint control (Deng et al., 1995; Brugarolas et al., 1995).These mice also did not show an increased incidence of tumors, suggesting that p21WAF1/C'P1is not important for the tumor suppressor function of p53 (Deng et al., 1995; Brugarolas et al., 1995). Another mechanism of p53 growth arrest in p21WAF1/C1P1-deficient mice may be the GADD45 gene, a transcriptional target of p53 (Kastan et al., 1992). GADD45 is a DNA damage-inducible gene that can cause growth arrests when overexpressed in cells (Zhan et al., 1994b). Another transcriptional target of p53 is cyclin G (Okamoto and Beach, 1994; Zauberman et al., 1995). Unlike GADD45, cyclin G overexpression enhances cell cycle progression and increases cisplatin sensitivity (Skotzko et al., 1995; Smith et al., 1997). The cyclin G protein has been shown to form a complex with the B' regulatory subunits of protein phosphatase 2A (PP2A) (Okamoto et al., 1996). Although the consequence of cyclin G interaction with the By subunits is not known, PP2A regulates many processes, including signal transduction, cell cycle, transcription, and development (for review see Mumby and Walter, 1993).

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B. Control of G2/M by p 5 3 Although p53 was originally thought only to control the Gl/S part of the cell cycle, several groups have shown that p53 may also function at the G2/M part of the cell cycle (Vikhanskaya et al., 1994; Stewart et a/., 1995; Agarwal et al., 1995; Aloni Grinstein et af., 1995).In addition, overexpression of the Ras oncogene in fibroblasts can also cause a G2/M arrest that is dependent on p53 (Hirakawa and Ruley, 1988; Hicks et d., 1991).p53 is also part of the spindle checkpoint control mechanism (Cross et al., 1995). Disruption of the mitotic spindle with drugs normally results in cell arrest at G2/M, but, in p53-deficient cells, multiple rounds of DNA synthesis occur without cell division (Cross et af., 1995).p53 also plays a role in controlling centrosome duplication (Fukasawa et af., 1996). Fibroblasts from p53-deficient mice show multiple copies of centrosomes that result in the abnormal segregation of chromosomes (Fukasawa et af., 1996). Alterations in the spindle checkpoint control and centrosome duplication in p53 mutant cells may play important roles in genetic instability seen in p53-deficient cells (Harvey et af., 1993; Tsukada et al., 1993). The growth arrest at G2/M caused by p53 overexpression may be mimicking the downstream events of the spindle checkpoint control. It is not yet clear whether p53 also functions as part of the G2/M checkpoint control due to unreplicated or damaged DNA. Cells deficient in p53 still show a G2/M arrest after irradiation (Kuerbitz et al., 1992). It is possible that more than one cell cycle checkpoint may be operational at G2/M (see later). How spindle damage activates p53 activity and how p53 controls the G2/M part of the cell cycle are unknown at this time.

C. Nontranscriptional Controls of Cell Cycle by p 5 3 It has become appreciated that nontranscriptional activities of p53 are also important for its cell cycle control. Transcription-defective mutants of p53 were previously shown to still cause growth arrest, suggesting the importance of a nontranscriptional activity of p53 (Sabbatini et al., 1995; Hansen and Braithwaite, 1996). The poly-proline region (PP) of p53 (amino acids 58-93) has been implicated in nontranscriptional control of cell growth (Walker and Levine, 1996).This region of several ProXXPro amino acid repeats can be binding sites for Src homology domain 3 (SH3) proteins, which are involved in tyrosine kinase signal transduction (Walker and Levine, 1996). Deletion of the PP region does not affect the transactivation activity of p53 (Walker and Levine, 1996). Transfection of a PP region deletion mutant into the H1299 and SAOS-2 cell lines reduces colony formation by 2fold compared with 10-fold for wild-type p53 (Walker and Levine, 1996). It has been shown that the PP region of p53 is essential for the growth arrest

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induced by the Gasl gene (Ruaro et al., 1997). The Gasl gene needs a transcription-independent function of p53 to induce growth arrest when ectopically expressed in cells (Del Sal et al., 1995). Growth arrest induced by overexpression of the nuclear c-Abl tyrosine kinase functions through binding to p53 protein (Sawyers et al., 1994; Goga et al., 1995). The proline-rich domain of c-Abl and not the SH3 domain of c-Abl is needed for binding p53 in vitro (Sawyers et al., 1994; Goga et al., 1995). Induction of growth arrest by radiation or genotoxic drugs needs c-Abl kinase activity and p53 binding, but not transactivation of the p21WAF1’C1P1gene (Yuan et al., 1996a,b). This growth arrest is associated with downregulation of cdk2 kinase activity (Yuan et al., 1996a,b). It is unknown whether Abl protein binds to the p53 PP region. Elevated expression of the p210BCR-Ab’gene in hematopoietic cells induces a G2/M arrest and protects cells from radiation and chemotherapeutic-induced apoptosis (Bedi et al., 1995). The Lyn tyrosine kinase also induces a G2/M arrest after the irradiation of hematopoietic cells (Kharbanda et al., 1994; Uckun et al., 1996). The X-irradiation of hematopoietic cells results in an inhibitory tyrosine 1 5 phosphorylation of cdc2 kinase that is dependent on Lyn kinase activity (Kharbanda et al., 1994; Uckun et al., 1996). It is not known if the Lyn kinase also needs p53 for its growth arrest. If Lyn or c-Abl tyrosine kinases are acting on cdc2 function without p53, this may explain why pS3deficient cells still show a G2/M growth arrest after irradiation.

1V. p53 AND APOPTOSIS A. p53 Control of Apoptosis in Tumors Wild-type p53 was initially shown to induce apoptosis by overexpression in myeloid leukemic and colon tumor cell lines (Yonish-Rouach et al., 1991; Shaw et al., 1992). The first evidence that the normal endogenous p53 could induce apoptosis under physiological conditions came from studies expressing the adenovirus E1A oncogene in the REF52 cell line and primary baby rat kidney (BRK) cells (Lowe and Ruley, 1993; Debbas and White, 1993). This E l A-induced apoptosis can be increased by the removal of serum. E1Ainduced apoptosis can be prevented by either coexpression of a dominantnegative mutant p53 or coexpression of the adenovirus E1B 19K or 55K proteins (Lowe and Ruley, 1993; Debbas and White, 1993). The E1B 55K protein binds p53 and inactivates its transactivation function (Yew and Berk, 1992). The E1B 19K protein, a viral homolog of the Bcl-2 oncogene, blocks apoptosis downstream of p53 (Boyd et al., 1994). Wild-type p53 is needed for apoptosis induction by ionizing radiation and

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some chemotherapeutic drugs (Clarke et al., 1993; Lotem and Sachs, 1993; Lowe et af., 1993; Lee and Bernstein, 1993). Thymocytes from p53 gene “knockout” mice demonstrate less radiation (Lowe et al., 1993; Clarke et af., 1993; Lotem and Sachs, 1993) or etoposide (Clarke et al., 1993)-induced apoptosis compared with normal mice. In contrast, lack of p53 does not affect apoptosis induced by synthetic glucocorticoids or Ca2+plus phorbol esters (Clarke et al., 1993; Lotem and Sachs, 1993). In the myeloid bone marrow progenitor cells from p53-deficient mice, increased survival was found at low concentrations of colony-stimulating factors and interleukins (Lotem and Sachs, 1993). Other studies have shown that expression of a dominantnegative mutant p53 in transgenic mice resulted in increased radioresistance in hematopoietic cells, but no difference in resistance to the alkylating agent ethyl methane sulfonate (Lee and Bernstein, 1993). Compared to thymocytes, primary fibroblasts from mice show reduced ionizing radiation or chemotherapeutic drug-initiated apoptosis (Lowe et al., 1993). Treatment of normal fibroblasts with ionizing radiation will normally cause growth arrest and not apoptosis (Kastan et al., 1992). Expression of an oncogene such as the adenovirus E1A is needed to sensitize normal fibroblasts to these treatments and E1A expressing fibroblasts from p53-deficient mice show resistance (Lowe et al., 1993). This resistance to y-irradiation and adriamycin treatments of p53 minus fibrosarcomas has also been demonstrated in vivo (Lowe et al., 1994a).These results lead to the prediction that drug resistance in the c h i c may sometimes be due to the loss of p53-dependent apoptosis in tumors. It has been found that p53 mutation is an independent predictor of clinical drug resistance in relapsed non-Hodgkin’s lymphoma (Wilson et al., 1997).The clinical aspects of p53 mutation and response to therapy have been reviewed by Ruley (1996).

B. p53 Transactivation and Apoptosis How p53 induces apoptosis is still not fully understood at this time. Several studies using various p53 transactivation-defective mutants have shown that induction of apoptosis by p53 is dependent on its transcriptional transactivating activity (Sabbatini et al., 1995; Yonish-Rouach et al., 1995; Ishioka et al., 1995; Attardi et al., 1996; Hansen and Braithwaite, 1996). One potentially important transcriptional target for p53-induced apoptosis is the Bax gene. Bax is a member of the Bcl-2 gene family that promotes cell death (for review see Reed et al., 1996). It has been proposed that cell death agonists of the Bcl-2 family, such as Bax, dimerize with Bcl-2 to promote cell death (Oltvai et a/., 1993). Overexpression of p53 protein or induction of p53 by DNA-damaging agents increases the expression of the Bax gene and decreases the expression of the Bcl-2 gene (Miyashita et al., 1994b; Zhan et

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al., 1994a). p53 DNA-binding sequences have also been found in the Bax gene promoter (Miyashita and Reed, 1995). Transfection of the Bax gene into BRK cell lines, expressing adenovirus E1A and mutant p53 genes, can reduce colony formation and induce apoptosis, supporting a role for Bax downstream of p53 (Han et al., 1996). In another study using E1A expressing fibroblasts from Bax-deficient mice, loss of the Bax gene resulted in partial drug resistance, although the resistance was not as great as p53-deficient fibroblasts (McCurrach et al., 1997). These results suggest that Bax may be part of a p53-dependent apoptosis pathway, depending on the tissue type, and that other factors controlled by p53 may still be needed for full apoptosis induction. Other studies have shown that Bax is not important for p53-dependent apoptosis in some tissues. For example, thymocytes expressing a Bax transgene showed increased levels of y-irradiation-, etoposide-, and dexamethasone-induced apoptosis (Brady et al., 1996). In contrast, expression of the Bax transgene in a p53 minus background did not increase DNA damageinduced apoptosis compared with p53+ thymocytes not carrying the Bax transgene (Brady et al., 1996). In another study, thymocytes from Bax-deficient mice showed similar levels of y-ray-induced apoptosis compared with normal litter mates (Knudson et al., 1995). In Epstein-Bar virus immortalized B cells and an interleukin-3-dependent leukemia cell line with a wildtype p53, there were no changes in the levels of Bcl-2 or Bax proteins after DNA-damaging agents (Shaulian et al., 1995; Canman et al., 1995). Expression of an exogenous Bax gene in E1A expressing p53-'- fibroblasts did not fully restore drug-induced apoptosis (McCurrach et al., 1997). Transfection of a wild-type p53 gene into Saos-2 and H1299 tumor cell lines does not result in any changes in Bax protein levels compared with nontransfected controls (Rowan et al., 1996). These contradictory roles for Bax in p53dependent apoptosis may be due to tissue-specific differences. Zn vivo p53 can alter the expression of Bax in neuronal, prostate, kidney, and small intestinal cell types, whereas Bax and Bcl-2 are not detected in cortical thymocytes (Miyashita et al., 1994b). Because Bcl-2 can block p53-dependent apoptosis (Wang et al., 1993; Wagner et al., 1993; Chiou et al., 1994; Wang et al., 1995c), even in cells without Bax (McCurrach et al., 1997), other death-inducing members of the Bcl-2 family may be possible targets of p53 transactivation, such as Bak and Bik (Boyd et al., 1995; Chittenden et al., 1995; Kiefer et al., 1995). Another transcriptional target of p53 that may be important in apoptosis induction is the insulin growth factor I-binding protein 3 (IGF-BP3) (Buckbinder et al., 1995). Insulin growth factor-1 (IGF-1) can suppress apoptosis induced by Myc oncogene overexpression (Harrington et al., 1994). An increased expression of IGF-BP3 will bind IGF-1 and, in turn, prevent binding to the IGF-1 receptor and promote apoptosis. Interestingly, the apoptosis-

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defective mutants p53Ala-143 and p53Pro-175 failed to activate properly the IGF-BP-3 and Bax gene promoters (Ludwig et al., 1996; Friedlander et al., 1996). The p53Leu-181 allele did not activate IGF-BP3 box B promoter sequences (Ludwig et al., 1996; Friedlander et al., 1996). All of these mutant p53s were still also able to induce growth arrest and activate the WAFZ\CIPI gene. The p53Ala-143 allele was previously shown to be temperature sensitive for growth, but defective for apoptosis induction (Kobayashi et al., 1995). A minority of the tumor-derived mutant p53s at position 175 still show wild-type levels of transcriptional and growth arrest properties (Ory et al., 1994; Crook et al., 1994). It was suggested that this differential induction of apoptotic versus growth arrest genes may reflect differences in the ability of the wild-type p53 protein to modulate expression of these genes, depending on the cellular context (Ludwig et al., 1996; Friedlander et al., 1996). Relevant to this idea is the finding that the substitution of arginine 175 on p53 to alanine will disrupt interaction of the p53-binding proteins 53BP1 and 53BP2, but not alter its transactivation or DNA-binding properties (Thukral et al., 1994; Iwabuchi et al., 1994). The fourth ankyrin repeats of 53BP2 make contact with this region on p53 and its SH3 domain to the loop 3 region of p53 (amino acids 241-248) (Gorina and Pavletich, 1996). It is not known whether the p53 tumor-derived apoptosis mutants Pro-175 and Leu-181 still bind 53BP1 or 53BP2 (Gorina and Pavletich, 1996). The temperature-sensitive mutant p53Ala-143 binds 53BP2 at the permissive temperature, but not at the restrictive temperature (Gorina and Pavletich, 1996). The 53BP2 protein has been shown to bind protein phosphatase 1 ( P P l ) and inhibit its activity (Helps et al., 1995).This interaction may result in changes in p53 phosphorylation and regulation (Helps et al., 1995). It remains to be determined whether the binding of 53BP1 or 53BP2 to p53 plays a role in the modulation of apoptotic versus growth arrest genes. It is interesting that the p53-binding protein 53BP2 has been shown to bind Bcl-2 protein (Naumovski and Cleary, 1996). Overexpression of 53BP2 in cells does not induce apoptosis but causes a G2/M arrest (Naumovski and Cleary, 1996). Another potential target for p53 transactivation in apoptosis is the Fas/APO-1 receptor that is upregulated by p53 (Owen-Schaub et al., 1995). Expression of wild-type p53 in a colon tumor cell line resulted in increased anti-Fas antibody killing (Tamura et al., 1995). Engagement of the Fas/APO1 receptor by the Fas ligand or anti-Fas antibody will induce apoptosis such as in activated T lymphocytes (Nagata and Golstein, 1995). Many tumor types have been shown to express the Fas ligand, and this has been suggested as a tumor defense against infiltrating cytotoxic lymphocytes that express Fas (O’Connell et al., 1996; Saas et al., 1997; Niehans et al., 1997). Loss of wild-type p53 and the resulting downregulation of the Fas/APO-1 receptor

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would then prevent death being induced by cells within the tumor that also express the Fas ligand or to infiltrating cytotoxic lymphocytes.

C. Nontransactivator Function in Apoptosis by p53 The first evidence for a nontransactivator role for p53 in apoptosis came from experiments in SV40 virus large T antigen-transformed somatotropic cell lines (Caelles et al., 1994). Ultraviolet induction of apoptosis was dependent on wild-type p53 activity in these cells and could not be blocked by inhibition of RNA or protein synthesis. In another study, protein synthesis inhibition or cell cycle arrest did not block Myc oncogene-induced apoptosis, which was p53 dependent (Wagner et al., 1994). A carboxyl-terminal deletion mutant of p53, which is transactivation defective, can still induce apoptosis in the HeLa cell line (Haupt et al., 1995b). Also, a p53 transactivation mutant, consisting of substitution of amino acids Glu22,Ser23 in the transactivation domain, would still induce apoptosis in HeLa cells (Haupt et al., 199%). In contrast, a temperature-sensitive variant of the p53(Glu22, Ser23-Va1135) mutant failed to induce apoptosis in baby rat kidney cells (BRK) transformed with the E1A oncogene (Sabbatini et al., 1995). This transactivation mutant of p53 still induced growth arrest, mostly in the S phase (Sabbatini et al., 1995). A human tumor-derived mutant p53prol75 retains sequence-specific transactivation and growth arrest, but was defective for apoptosis induction and suppression of transformation by papilloma virus E 7 and Ras oncogenes (Rowan et al., 1996). These results suggest that the transactivation and induction of apoptosis functions of p53 can be separated genetically. It has been argued that the induction of apoptosis by transactivation-defective p53 mutants may be due to the presence of an endogenous wild-type p.53 in the cell lines used (Attardi et al., 1996). The mutant p53 may bind to the papilloma virus E6 protein in HeLa cells to release wild-type p53 from degradation. Alternatively, the mutant p53 may bind with the endogenous wild-type p53 and allow transactivation. Coexpression of the p53-binding protein mdm2, which inhibits p53 transactivation function, failed to block apoptosis induced by wild-type p53 in HeLa cells, although p53 transactivational activity was blocked (Haupt et al., 1996). mdm2 can block apoptosis in cell lines that require p53 transactivation to induce apoptosis (Haupt et al., 1996; Chen et al., 1996). This argues against the mutant p53 competing for binding to the E6 protein in HeLa cells and releasing the endogenous wild-type p53 protein. Transfection of the p53Gln22,Ser23 mutant into a cell line expressing wild-type p53 results in transactivation of a reporter gene containing a p53 promoter (Roemer and Mueller-Lantzsch, 1996).

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However, the p53Gln22,Ser23 mutant will induce a delayed apoptosis in the p53 minus Saos-2 cell line (Chen et al., 1996).A p53 carboxyl-terminal deletion mutant, defective for transactivation, will also inhibit colony formation in the Saos-2 cell line (Ishioka et al., 1995).p53 induces both apoptosis and growth arrest in Saos-2 cells (Marcellus et al., 1996; Rowan et al., 1996). Therefore, p53 transactivation-dependent and -independent pathways may both be needed for full apoptosis induction. The rate of apoptosis induced by the wild-type p53 is greater than the rate induced by the p53 transactivation mutants alone (Haupt et al., 1995b). Overall, these results suggest that, depending on the cell type and genetic background, the transactivating function of p53 may be needed for apoptosis induction. In other cell types already primed to undergo apoptosis, a nontransactivation function of p53 is sufficient. One possible activity that may play a role in apoptosis is the transcriptional repressor activity of p53. Blocking p53-dependent apoptosis by coexpression of adenovirus E1B 19k or Bcl-2 proteins results in the enhancement of p53-mediated transactivation and inhibition of the transcriptional repressor activity of p53 (Shen and Shenk, 1994; Sabbatini et al., 1995). It is not yet known how Bcl-2 or E1B 19K proteins inhibit the repressor activity of p53. Another study using various p53 mutants showed that the growth suppressing activity of p53 can be genetically separated from its apoptotic functions (Hansen and Braithwaite, 1996). This study also found that the transcriptional activation and repressor activities of p53 are both necessary for apoptosis induction. The Wilms’ tumor suppressor gene product binds p53 and blocks apoptosis (Maheswaran et al., 1995).This binding results in an increase in p53 transactivational activity, but reduces its repressor activity (Maheswaran et al., 1995). What are the target genes for p53 repression of transcription in apoptosis induction? p53 has been shown to negatively regulate the expression of the anti-apoptotic Bcl-2 gene (Miyashita et al., 1994a; Haldar et al., 1994).The 5’-untranslated region of the Bcl-2 gene is important for this p53-dependent repression (Miyashita et al., 1994a). Another target for the repressor activity of p53 is the IGF-1 receptor gene (Werner et al., 1996; Prisco et al., 1997). Loss of p53 activity through mutation results in increased IGF-1 receptors and protects tumor cells from apoptosis through IGF-1 binding to its receptor (Werner et al., 1996; Prisco et al., 1997). The P3 promoter of IGF-I1 is also inhibited by wild-type p53 (Zhang et al., 1996). Wild-type p53 suppresses the expression of the microtubule associated protein 4 gene (MAP4) in cells undergoing apoptosis (Murphy et al., 1996).Expression of the MAP4 gene in cells delays the onset of apoptosis (Murphy et al., 1996). Other studies using p53 mutants defective in transcriptional repressor activity have not shown a correlation with apoptosis. The transactivation-defective mutant p53 Gln22,Ser23 also has impaired repressor activity (Roe-

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mer and Mueller-Lantzsch, 1996), yet it still induced apoptosis in HeLa cells (Haupt et al., 199513). Fusion of the VP-16 transactivator domain to p53 amino acids 100-393 results in a p53 that is defective for transactivation but still able to repress transcription (Pietenpol et al., 1994). The VP-16 p53 fusion protein does not induce apoptosis in E l A-transformed fibroblasts (Attardi et al., 1996). In contrast, a transcription competent fusion mutant VP16 (80-393)~53could induce apoptosis (Attardi et al., 1996). Another p53 mutant could repress transcription and cause growth arrest, but was defective for apoptosis induction (Hansen and Braithwaite, 1996). These results suggest that repression of transcription by p53 may not be sufficient for apoptosis induction, but may enhance apoptosis under certain circumstances. Support for this view has been shown in a study using various p53 mutants that found that both transactivation and repression functions may be needed for apoptosis induction (Hansen and Braithwaite, 1996). It has been suggested that the interaction of p53 with XP-B and XP-D helicases that are members of the TFIIH transcription factorhepair complex may also play a role in apoptosis induction (for a review see Warbrick, 1996). p53 also binds to another member of the TFIIH complex, p62 (TFB1) (Xiao et al., 1994; Leveillard et al., 1996). TFIIH is a general transcription factor complex containing helicase and kinase activities (for a review see Orphanides et al., 1996). Some members of the TFIIH complex are the gene products of the human hereditary DNA repair disorders xeroderma pigmentosum (XP) and Cockayne’s syndrome (CS) (Seroz et al., 1995). Also associated with TFIIH is cyclin-dependent kinase 7 (CDK7)and its partner cyclin H, which is responsible for phosphorylating the carboxyl-terminal domain of RNA polymerase I1 (for a review see Nigg, 1996). It is also responsible for activating cyclin-dependent kinases (CAK),such as cdkl kinase (cdc2). In addition to its role in transcription, the TFIIH complex plays an important role in DNA excision repair, allowing the incision step to continue at the DNA lesion (Sancar, 1996). Both mutant and wild-type p53 proteins bind to the ERCC3 (XPD)and ERCC2 (XPB)helicases and inhibit both 5‘-3’ and 3’-5’ helicase activities in vitro (Wang et al., 1994; Leveillard et al., 1996). Fibroblasts from patients with mutations of the XP-D and XP-B genes showed reduced apoptosis after injection of p53 expression vectors compared with normal fibroblasts (Wang et al., 1996). This apoptosis-defective phenotype can be rescued by coexpression of the wild-type XP-B and XP-D genes (Wang et al., 1996).The XP-D and XP-B mutations did not prevent p53 transactivation of the WAFlICIPl gene (Wang et al., 1996). It is possible that the interaction of p53 with the TFIIH complex may influence the control of expression of apoptosis-inducing genes such as Bax or ZGFlBP3, which appear to have a different level of control. It has been shown that TFIIH will relieve p53-mediated transcription inhibition at the IgH promoter in uitro (Leveillard et d., 1996).

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The carboxyLterminal26 amino acids of p53 are needed for binding XPD and XP-B (Wang et al., 1995b), and microinjection of this region of p53 into cells can induce apoptosis (Wang et al., 1996). In contrast, transfection of a p53 gene containing only the first 214 amino acids into HeLa cells can still induce apoptosis (Haupt et al., 1995b). The alternatively spliced form of p53, which has the XP-D- and XP-B-binding domain removed, induces apoptosis, but with slower kinetics compared with the regularly spliced form of p53 (Almog et al., 1997). The effect of p53 interaction with TFIIH on excision repair is not clear at this time. Consistent with the inhibition of p53 of the XP-D and XP-B helicases, fibroblasts from Li-Fraumeni patients heterozygous for a p53 codon 145 mutation showed reduced repair of pyrimidine dimers (Wang et al., 1995b). Another study using Li-Fraumeni syndrome fibroblasts found that loss of p53 did not affect excision repair of pyrimidine dimers from the transcribed stand (transcription-coupled repair) (Ford and Hanawalt, 1995). p53-deficient cells showed a reduced removal of dimers from the nontranscribed strand, which is indicative of a global defect in excision repair (Ford and Hanawalt, 1995). This phenotype resembles xeroderma pigmentosum complementation group C (XP-C)cells (Venema et al., 1991; van Hoffen et al., 1995). Crosses of XP-C and p53-deficient mice result in a synergistic increase in severe solar keratosis and increased skin cancer after UV irradiation (Cheo et al., 1996).These mice also showed a variable spectrum of neural tube abnormalities (Cheo et al., 1996). This genetic interaction of p53 and XP-C suggests that they may be acting at a common DNA lesion or that the proteins may be in direct contact in the repair complex. The XP-C protein, along with its partner HHR23B, is needed for excision repair of pyriniidine dimers but not bulky DNA lesions in vitro (Mu et al., 1996). In transcribed DNA, the XP-C protein may not be needed for excision repair of lesions next to a stalled RNA polymerase (Sancar, 1996; Mu et al., 1996). Because of its nonspecific binding to single-stranded DNA, it was suggested that XP-C may play a role in stabilizing unwound DNA for incision and protecting the damaged DNA from nuclease digestion (Mu et d., 1996; Sancar, 1996).Interestingly, fibroblasts mutant for CS-A, CS-B, and XP-A, which are defective in preferential repair of the transcribed strand, show increased UV induction of p53 and apoptosis compared with XP-C and normal fibroblasts (Ljungman and Zhang, 1996). The stalling of RNA polymerase at DNA lesions was suggested as an important trigger for p53-dependent apoptosis induction (Ljungman and Zhang et al., 1996). It is not known whether p53 binding to the TFIIH repair complex at the stalled RNA polymerase complex plays a role in this induction. It has also been reported that Li-Fraumeni fibroblasts show a defect in long-patch excision repair after exposure to UV or y-radiation (Mirzayans et al., 1996). Other studies have failed to show a direct inhibition of excision repair by

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pS3 in uitro assays that is dependent on TFIIH activity (Leveillard et al., 1996; Sancar, 1996).Fibroblasts from p53 knockout mice do not show any differences in the removal of UV-induced pyrimidine dimers or (6-4) photo products compared with normal mice, but do show an increase in sister chromatid exchange (Ishizaki et al., 1994). Although p53 may not be directly involved in excision repair, its association with members of the TFIIH complex may play associated roles in DNA repair, such as repair of nontranscribed regions of the genome. More work is still needed to clarify the role of p.53 interaction with the TFIIH complex with respect to DNA repair, transcription, and apoptosis. Another region of pS3 implicated in nontranscriptional control of apoptosis by p.53 is the poly-proline region. Deletion of the PP region in the mouse pS3 gene is critical for the induction of apoptosis in E1A expressing cells (Sakamuro et al., 1997). This deletion does not affect the ability of pS3 to genes (Sakamuro et al., 1997). The inability of induce Bax or p21WAF1’CrP1 a p.53 transactivation region deletion mutant (AA 11-69) to induce apoptosis in HeLa cells (Yonish-Rouach et al., 1995) may be due to the partial removal of the PP region. These results show that a nontranscriptional activity of p53, presumably through binding to an SH3 containing protein, is necessary for pS3 induction of apoptosis and that transcription may be necessary but not sufficient for apoptosis induction. The identity of this protein(s) is unknown at this time. Whatever the mechanisms of p53-induced apoptosis, the events downstream from pS3 result in activation of the ZCEICed3 family of caspase proteases to manifest the late events in apoptosis (Sabbatini et al., 1997).

V. p53 AND TUMOR PROGRESSION

A. Apoptosis and Tumor Progression It is now accepted that tumor cell progression results from the accumulation of multiple mutations in both tumor suppressor genes and oncogenes. These mutations occur randomly, but to be selected for they must give a growth advantage to the tumor cell. The increased expression of many oncogenes or inactivation of tumor suppressor genes can make a cell susceptible to apoptosis that is dependent on p53. Expression of the adenovirus E1A oncogene (Lowe and Ruley, 1993; Debbas and White, 1993), inactivation of the retinoblastoma (Rb) gene by mutation or binding to the papilloma virus E7 protein (Morgenbesser et al., 1994; Pan and Griep, 1994; Almasan et al., 1995), expression of the E2F transcription factor (Ramqvist et al., 1993; Wu and Levine, 1994; Qin et al., 1994), and M y c oncogene expression

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(Ramqvist et al., 1993; Hermeking and Eick, 1994) can increase apoptosis in cells that can be attenuated by p53 inactivation. These genes either disrupt or bypass the RbE2F complex that controls GUS cell cycle progression (for review see Harper and Elledge, 1996). Release of the E2F transcription factor from the phosphorylated Rb protein allows activation of cell cycleregulated genes such as c-myc, dihydrofolate reductase, and thymidylate synthase (Lam and La Thangue, 1994). Consistent with this view is the finding that overexpression of the Rb protein can induce cell cycle arrest and prevent p53-dependent apoptosis (Qin et af., 1994; Haupt et af., 1995a). Disruption or Rb function allows cells to overcome p53 G1-dependent growth arrest (Slebos et al., 1994). It should also be noted that oncogene and Rbdependent apoptosis can also occur by a p53-independent mechanism (Teodoro et a1.,1995; Sakamuro et al., 1995; Macleod et al., 1996). The ability of p53 to prevent oncogene-mediated apoptosis was shown as an important selective advantage for tumor progression (Lowe et al., 1994b). Cells containing the E1A and Ras oncogenes in a wild-type p53 background still formed tumors in nude mice, despite showing increased apoptosis (Lowe et al., 1994b). Absence of p53 in these tumors resulted in shorter tumor latency. Loss of p53 in an in vivo choroid plexus tumor model induced by a truncated large T antigen that binds Rb protein but not p53 resulted in aggressive tumor outgrowths and reduced apoptosis (Symonds et af., 1994).In this tumor model, crossing of truncated large T antigen mice to Bax-deficient mice resulted in a more rapid tumor growth rate and reduced overall survival (Yin et al., 1997).However, this effect was not as great as seen in p53deficient mice. The apoptotic index was reduced by approximately 50% in CP tumors from Bax-deficient mice compared to 90% in tumors from p53deficient mice (Yin et af., 1997). These data suggest that Bax may be responsible for about 50% of the p53-dependent apoptosis seen in these tumors, but that other p53 targets are also important for apoptosis. These data support the idea that p53 inactivation is not essential for tumor growth, but that loss of p53 apoptotic function results in more aggressive tumor outgrowths in cells with oncogene activation or tumor suppressor loss. Environmental factors can also alter the response of a transformed cell to apoptotic signals. The presence of growthhurvival factors can greatly reduce p53 and oncogene-mediated apoptosis (Yonish-Rouach et al., 1991; Collins et af., 1992; Canman et af., 1995; Lin and Benchimol, 1995b).Transfection of oncogenes on growth factor signal transduction pathways such as Src, Ras, and Raf can rescue cells from p53-dependent apoptosis (Cleveland et al., 1994; Lin et al., 1995a; Canman et al., 1995). Interestingly, although oncogenes such as Ras can overcome apoptosis, it cannot overcome p53 growth arrest without the coexpression of the E l A gene (Lin et af., 1995a). Rat fibroblasts can be transformed with the papilloma E 7 and Ras oncogenes with or without p53 mutation (Peacock and Benchimol, 1994). Clones with

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a mutated p53 gene can grow in low serum conditions and respond to autocrine growth factors. In contrast, transformed cells with a wild-type p53 gene need external growth factors and do not respond to autocrine factors (Peacock and Benchimol, 1994). This difference in response to autocrine or external growth factors by tumors may be an important selective pressure for loss of p53 in vivo. Hypoxic conditions within the tumor may be another important environmental trigger for loss of p53 activity. p53 is induced by hypoxic conditions, and transformed cells lacking p53 are more resistant to hypoxia-induced apoptosis (Graeber et al., 1996).

B. Growth Control and Tumor Progression Another possible selection for loss of p53 in tumors is to overcome growth arrest caused by oncogene overexpression. Previous work had shown that the elevated Ras oncogene expression in the REF52 cell line induces a block in the cell cycle at the GUS and G2/M boundaries that can be overcome by large T antigen expression (Hirakawa and Ruley, 1988; Franza, J . et al., 1986). This Ras growth arrest was also associated with an accumulation of neutral lipids within cells (Hirakawa et al., 1991). Similarly, in Schwann cells, transfection of Ras can cause growth arrest at GUS and G2/M, and the SV40 large T antigen can overcome this growth arrest (Ridley et al., 1988). It has been shown previously that inactivation of p53 function through transfection of dominant-negative mutant p53 allows elevated Ras expression and increased tumorigenicity in REF52 cells (Hicks etal., 1991).In another study using mouse primary prostate cells, transfection of the Ras oncogene always resulted in selection for p53 mutations (Lu et al., 1992). In contrast, transfection of Myc and Ras oncogenes induced the formation of carcinomas that expressed elevated levels of the wild-type p53 protein (Lu et al., 1992).These Myc/Ras cells also showed evidence of increased apoptosis. Another study showed that transformation by EJ-Ras is susceptible to wild-type p.53 suppression (Hansen et al., 1995). It was also in this study that the function of large T antigen responsible for overcoming p53 suppression of transformation was the Rb-binding. In a MMTV-Ras transgenic mouse model, crosses to p53-deficient mice showed an increase in salivary tumors without an alteration of apoptosis rates, but increased genomic instability compared with Raslp53”’ mice (Hundley et al., 1997). It has been shown that Ras oncogene expression can induce growth arrest and premature senescence in primary fibroblasts (Serrano et al., 1997). This premature senescence could be overcome by inactivation of either p53 or cdk4 inhibitor ~ 1 6 in” rodent ~ ~cells. ~ In human primary cells, neither p53 nor ~ 1 6 inactivation ” ~ ~ was ~ sufficient to overcome the Ras growth arrest. However, E1A oncogene coexpression could overcome Ras-induced growth

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arrest in human cells (Serrano et al., 1997).It has also been shown that overexpression of the Mos oncogene and members of the MAP kinase pathway, including activated Ras, Raf, and M E K oncogenes, induce growth arrest and apoptosis in fibroblasts (Fukasawa and Vande Woude, 1997). This lethality was greatly reduced in fibroblasts from p53-I- mice. In primary rat Schwann cells, Raf oncogene expression induces growth arrest that is mediated by p21wA"1'c1p1, which can be overcome by mutant p53 or large T antigen (Lloyd et al., 1997). Collectively, these studies strongly suggest that there is a strong selective pressure to inactivate p53 in a cell that overexpresses activated members of the RasIMAP kinase pathway. This Ras-induced growth arrest resembles the premature senescence seen in normal diploid fibroblasts induced by y-radiation that is dependent on p53 (Di Leonardo et al., 1994; Linke etal., 1997).Radiation-induced growth arrest is presumably due to unrepaired double strand breaks causing p53 stabilization and p21WAF1/C1P1 induction or other p53-dependent factors (Di Leonardo et al., 1994; Linke et al., 1997). Induction of the cdk inhibitor p21WAF1/C1P1 (SDIl) was previously associated with senescent cells (Noda et al., 1994). It has been hypothesized that telomere erosion seen in aging cells may trigger p53 growth arrest (Bond et al., 1994; Wynford-Thomas et al., 1995). p53 arrest can be reversible when it is induced by a decrease in ribonucleotide pools or when using an inducible system (Agarwal et al., 1995; Linke et al., 1996). This suggests that p53 may not be inducing senescence directly, but that it is important for permanent growth arrest. In cases of unrepaired DNA breaks or constitutive Ras oncogene expression, this arrest becomes permanent, leading to senescence unless p53 is inactivated. It is not known at this time if Ras induces DNA breaks directly. However, several studies have shown that induced expression of members of the Ras/MAPK pathway can lead to genomic instability and apoptosis (Denko et al., 1994, 1995; Fukasawa and Vande Woude, 1997). Alternatively, high levels of constitutively activated Ras may elevate p53 or p161NK4activity directly. As shown previously, pS3 can be phosphorylated by MAP kinase (Milne et al., 1994). However, in normal nonimmortal cells, Ras signaling functions to cause phosphorylation of Rb protein and E2F release by altering cyclin Ddependent kinases and p161NK4(Peeper et al., 1997; Mittnacht et al., 1997). Understanding how high levels of constitutively activated Ras leads to p21WAF1/C1P1 and p 161NK4expression and growth arrest will be important for understanding the basis of oncogene cooperativity. The possible pathways for p53 tumor suppression are shown in Fig. 2.

C. Genomic Instability and Tumor Progression Germline inactivation of p53 on its own can also result in spontaneous tumors in mice or Li-Faumeni patients (Donehower et al., 1992; Jacks et al.,

p53 in Tumor Progression

41

Fig. 2 Possible pathways for the induction of the tumor suppressor function of pS3. Input signals to p53 are shown in italics and downstream pathways are shown in bold. Various inputs from activated oncogenes and DNA damage to hypoxia can activate p53 function to cause either growth arrest and senescence or apoptosis.

1994; Malkin, 1994). Spontaneous mutation of oncogenes in a cell without a functional p53 gene would have an automatic growth advantage compared with a cell with a wild-type p53 gene by the previously mentioned mechanisms. However, inactivation of p53 may also directly play a role in tumor progression through its role in controlling genomic stability. Loss of p53 has been previously associated with increased gene amplification (Yin et al., 1992; Livingstone et al., 1992). Increased expression and amplification of the Met and Myc oncogenes were seen in tumors and primary cells from p53deficient mice and Li-Fraumeni patients (Tainsky et al., 1995; Rong et al., 1995; Fukasawa et al., 1997). The increased aneuploidy found in pS3-Icells could reveal other tumor suppressor mutations through chromosome loss or increased oncogene expression due to a gain in chromosome numbers. The increased propensity for gene amplification seen in p53-deficient cells may also increase the likelihood of amplification of drug-resistant genes following chemotherapeutic treatment of tumors (Yin et al., 1992; Livingstone et al., 1992; Perry et ul., 1992).

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As knowledge of the p53 status in tumors becomes more routine in the clinic, knowledge of the context in which p53 mutations are found will be important. Depending on the order of oncogene activation, p53 may have been selected to prevent apoptosis or senescence in the tumor, which in turn may influence the response of the tumor to certain therapies. Therefore, the context in which pS3 mutations occur will be important to understand. Also, the many genetic variants within the tumor may lead to outgrowths of drugresistant or metastatic clones. It remains to be seen whether the knowledge of p53 status in tumors can be successfully exploited for treatment and if this will also lead to the discovery of new treatments or strategies. More research is still needed to give answers to these questions.

ACKNOWLEDGMENTS I thank my colleagues Sabine Mai, Jennifer Brown-Gladden, Dan Gietz, and Arnold Greenberg for comments and reading the manuscript. I also thank George Prendergast, Eileen White, and Sabine Mai for sharing papers before publication and Steve Linke for discussions via e-mail.

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Signal Transduction through MAP Kinase Cascades Timothy S. Lewis, Paul S. Shapiro, and Natalie G. Ahn Department of Chemistry and Biochemistry, Howard Hughes Medical Institute University of Colorado, Boulder, Colorado 80309

I. The MAP Kinase (MAPK) Module 11. Mammalian MAPK Pathways A. ERKlR and MKKlR Pathways B. Stress-Activated Protein Kinase Pathways 111. Regulation of MAPK Pathways by Protein Phosphatases A. Dual Specificity Phosphatases B. Serinenhreonine Phosphatases C. Protein Tyrosine Phosphatases IV. Cellular Substrates of MAP Kinases A. Protein Kinase Substrates for MAPKs B. Nuclear Transcription Factors C. Signaling Components D. Cytoskeletal Proteins V. Responses to MAPK Pathways: Growth and Differentiation A. Regulation of Cell Growth and Transformation B. Regulation of Cell Differentiation and Development VI. Yeast MAPK Pathways A. Saccharomyces cereuisiae (Budding Yeast) B. Schizosaccharomyces pombe (Fission Yeast) VII. Intracellular Targeting and Spatial Regulation of MAPK Pathway Components A. Signaling Complexes B. Nuclear Translocation of MAPK and MKK VIII. Future Directions References

I. THE MAP KINASE (MAPK) MODULE The identification of MAP kinase pathways exemplifies the power of combining biochemical and genetic approaches to molecular problems. Components in these pathways were first discovered in genetic studies of the pheromone-regulated mating response in Saccharomyces cerevisiae as genes that showed homologies with protein kinases (Errede and Levin, 1993). Although these enzymes could be ranked genetically, evidence of their protein kinase activities and the realization that they functioned in the context of a Advances In CANCER RESEARCH

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kinase cascade awaited biochemical studies of growth regulation in mammalian cells. As a result of earlier work establishing the importance of protein phosphorylation in cyclic nucleotide and calcium and phospholipid-regulated pathways, studies carried out during the 1970s focused on identifying kinases mediating growth factor and hormone action, particularly insulin. A key finding was the observation of growth factor-dependent phosphorylation of ribosomal protein S6 implicating factor-stimulated protein kinases in growth regulatory pathways. Combined with the discovery of tyrosine phosphorylation and receptor tyrosine kinases around 1980, protein kinase cascades in growth regulation were anticipated based on the paradigm of hormone regulated kinase cascades in the regulation of glycogen breakdown. Biochemical studies on hormone signaling in mammalian cell growth and oocyte maturation utilized strategies of cell stimulation followed by screening for regulated protein kinase activity. From these experiments, growth factor-stimulated pp90 ribosomal S6 kinases (Rsk)as well as MAP kinases, now known as ERKl and ERK2, were identified (Erikson and Maller, 1986; Ray and Sturgill, 1987; Jones et al., 1988; Hoshi et al., 1988; Ahn et al., 1990; Boulton et al., 1990), and the enzyme-substrate relationship between ERKs and Rsk was established (Sturgill et al., 1988; Ahn and Krebs, 1990). This was soon followed by biochemical identification of an upstream kinase regulator of ERK, called MAP kinase kinase (MKK), also known as MAPERK kinase (MEK) (Ahn et al., 1991; Gomez and Cohen, 1991). Molecular cloning of ERKsl/2 (Boulton et al., 1990, 1991) and MKKl (Crews et al., 1992; Kosako et al., 1993) established the homology of these enzymes with yeast FUS3KSSl and STE7, respectively, followed by confirmation that STE components were also regulated by direct phosphorylation. Homologous pathways have been identified in all eukaryotic organisms inspected so far. In nearly every case, a MAP kinase homolog regulated by a MKK homolog has been identified, thus the term “MAPK module” was coined to refer to the MAPIUMKK pair (Errede and Levin, 1993). At this point, evidence exists for at least three pathways involving MAPK modules in mammalian systems, five pathways in cerevisiae, and three pathways in Schizosaccaromyces pornbe (Figs. 1and 2). Functional MAPK modules have also been identified and characterized in frogs, fruit flies, nematodes, slime mold, and plants.

11. MAMMALIAN MAPK PATHWAYS In mammals, 11 distinct MAPK and 7 MKK genes have been identified to date. Members of the MAPK family include (i) extracellular signal-regulat-

cdl Growth and Differentiation

Stress Responses

Undefined

MEKK

MEK MAPK

\ J Mitosis. Meiosis. Diflerentiation, Development

I

1

1

Inflammation, Apcptosis

?

?

Fig. I Mammalian MAPK pathways. Positive signaling events are represented by solid pointed arrows (1)and inhibitory signaling is represented by blunted arrows (1) Signaling . events that are indirect or mechanistically uncharacterized are denoted by dashed arrows.

S. pornbe

S. cerevisiae Pheromone Response

Pseudohyphal or lnvasive Growth

a or a Factor

Nitrogen Starvation

High Osmolarity High Salt

Cell Wall Integrity

SpoNlatiOn

Hypotonic Shock Nitrogen Heat Shock Starvation Polarized Growth

Pheromone Response P or M

Stress Response

Cell Wall Integrity

Osmotic Stress Oxidative Stress

?

t

Cell Cycle Arrest

Mating Specific Genes

Filamentous Growth Genes

t

Osmotic Shock Genes

Cell Wall Biosynthesis Genes

Late Sporulation Genes

Mating, Stress Response, Cell Wall Sporulation Mating Specific Biosynthesis Genes Genes Genes?

Fig. 2 MAPK pathways of the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Positive signaling is represented by The . dashed line signifies a protein-protein insolid pointed arrows ( 1) and inhibitory events are denoted by solid blunted arrows (1) teraction between STES and G, (STE4). The dashed pointed and blunt arrows in the HOG pathway represent inhibited two-component signaling among SLN1, YPD1, and S S K l as caused by high extracellular salt concentrations.

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ed kinases (ERKs) 1 and 2, (ii)NH,-terminal Jun kinasehtress-activated protein kinases (JNWSAPK) a, p, and y, and (iii) p38 MAPKs a,p, y, and 8. These are specifically recognized and phosphorylated by: (i) MKKl and MKK2 (also known as MEKs 1 and 2), (ii) MKK4 and MKK7, and (iii) MKK3 and MKK6, respectively. Less well characterized is ERK3, for which upstream regulators have not been identified, ERK4, a protein immunocrossreactive with antibodies to ERKs 1and 2, and ERK5 (also known as big molecular weight kinase, BMK), which associates with MKKS. MAPKs and MKKs are closely related to each other by sequence, suggesting that these multicomponent kinase cascades arose through gene duplication of the MAPK module. MAPKs are activated by MKKs through common mechanisms involving phosphorylation at two regulatory phosphorylation sites with sequence T(P)-X-Y(P) located in the “activation lip” between subdomains 7 and 8 of the conserved kinase core sequence. Thus MKKs fall within a relatively rare class of protein kinases with dual specificity toward Ser/Thr and Tyr residues on exogenous substrates. MKKs are also activated by phosphorylation at Ser/Thr residues within the activation lip. Unlike MAPKs, which are specifically recognized by their corresponding MKKs, each of the MKKs can be phosphorylated and activated by several different MAP kinase kinase kinases (MKKKs), including Raf family members, C-MOS,MEK kinases (MEKKs), and multilineage protein kinases (MLKs).These MKKKs recognize different MKKs, enabling diversity in the activation of MAPK pathways upstream of MKK. As required for switching mechanisms, MAPK cascades transform graded effector signals into cooperative responses. This is a theoretical consequence of pathways involving sequential protein kinases, each regulated by dual phosphorylation, as demonstrated by Huang and Ferrell (1996b).

A. ERK1/2 and MKK1/2 Pathways ERKl and ERK2 and their upstream regulators MKKl and MKK2 are acutely stimulated by growth and differentiation factors, in pathways mediated by receptor tyrosine kinases, heterotrimeric G protein-coupled receptors, or cytokine receptors. At the moment, it is unclear why two forms of each enzyme exist, although conservation of two forms throughout eukaryotic species suggest nonredundant functions. These enzymes are expressed ubiquitously in mammalian cells at micromolar levels (Huang and Ferrell, 1996b), although some variation in expression between different tissues has been noted (Boulton and Cobb, 1991; Moriguchi et al., 1995a). 112 vitro, MKKl and MKK2 show comparable activity toward ERKl and ERK2 (Zheng and Guan, 1993a; Dent et al., 1994). A few examples exist where

Timothy S.Lewis et al.

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ERKl and ERK2 are activated with distinct magnitudes or kinetics following factor stimulation of intact cells (Papkoff et al., 1994; Kalb et al., 1996); however, in most instances, both enzymes are robustly activated on cell stimulation. 1.

ERKl AND ERK2

ERKl and ERK2 are 44- and 42-kDa enzymes, respectively; in mammals, these have 90% sequence identity (Boulton et al., 1991). In addition to the conserved catalytic core, both enzymes contain C-terminal extensions and a 25 amino acid insert between subdomains 9 and 10. Both enzymes are activated by phosphorylation within their activation lip at Thrl8,-Glu-TyrI8, (numbered as in rat ERK2) in a reaction that is partially ordered with Tyr preceding Thr phosphorylation (Haystead et al., 1992). Both phosphorylation events are required to fully activate wild-type ERKl and ERK2, although 10% activation can be achieved with mutants containing a Thr18,-Glu amino acid substitution, presumably due to the substitution of this phosphorylation site with a negatively charged residue (Robbins et al., 1993).Activation by phosphorylation leads to increased V, and decreased Km for peptide substrate, with little effect on KmATp(Robinson etal., 1996b). The crystal structure of ERK2 in its inactive, unphosphorylated form was solved by Goldsmith and colleagues to 2.3 A resolution (Zhang et al., 1994). Similarities within the conserved kinase domain to the structures of other protein kinases were observed, with the inclusion of an additional P-sheet structure at the N-terminal lobe (residues 1-20), a novel linker between p4 and PS (residues 91-96), and an a-helical insert between helices G and H in the C-terminal lobe (residues 247-266). Thirty-five C-terminal amino acids wrap in a random coil spanning the C-terminal to N-terminal lobes on the side of the molecule opposite to the catalytic cleft. Recombinant C-terminal residue truncations of ERK2 are poorly expressed (J. Means and N. Ahn, unpublished observations), suggesting that this region may play a role in enzyme stabilization. The two domains of ERK2 are rotated into an open configuration with respect to each other, and the activation lip is distorted with access to the catalytic cleft blocked by the Tyrlss phosphorylation site, thus providing an understandable rationale for the low basal activity. In this state, TyrlS5 is buried whereas Thr,,, is solvent accessible, suggesting that MKK binding induces a major conformational change in ERK to expose and enable preferential phosphorylation of the Tyr residue. Interestingly, substitution of Tyrls5 with acidic amino acids led to marked disorder within the activation lip (Zhang, F. et al., 1995). A model to account for these observations postulates induced fit binding of MKK and initial TyrlFs phosphorylation on ERK followed by substantial reconfiguration of the lip on Thr,,, phosphorylation. This stabilizes interactions between phosphorylated

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residues and catalytic site residues that favor the active conformation (Zhang, F. et al., 1995). Crystallographic determination of the activated ERK2 phosphorylated at Thr,,, and Tyrl,, supports this prediction by showing a reorientation of the activation lip, with phosphorylated Thr, bridging residues from both N- and C-terminal lobes and phosphorylated Tyr,,, forming the substrate binding pocket (Canagarajah et al., 1997).

,,

2. M K K l

AND MKK2

MKKl and MKK2 are both 44-kDa enzymes, related by 80% sequence identity (Zheng and Guan, 1993a; Wu et al., 1993a). Sequences outside the conserved catalytic core of both enzymes include an additional 60 amino acids at the N terminus and a 40 amino acid insert between subdomains 9 and 10. Activation occurs through phosphorylation at the sequence Ser,,8(P)-Met-Ala-Asn-Ser222(P) (numbered as in human MKKl ) within the activation lip (Zheng and Guan, 1994a; Alessi et al., 1994; Gotoh et al., 1994; Resing et al., 1995). Unlike ERKs, MKKs can be partially activated by phosphorylation at either serine phosphorylation site (Gotoh et al., 1994; Resing et al., 1995). In addition, substitution of these sites by acidic amino acids enhances the basal activity (Zheng and Guan, 1994a; Alessi et al., 1994; Cowley et al., 1994; Pages et al., 1994; Mansour et al., 1994a). Further enhancement of activity on substituting adjacent nonphosphorylatable residues with acidic amino acids indicates that the activation can be, in part, ascribed to electrostatic effects of the negatively charged residues (Mansour et al., 1996a). The N terminus of MKK outside the conserved kinase subdomain appears to be important in regulating MKK activity, as deletion of residues 44-51 or proline substitution within this region leads to significant activation (Mansour et al., 1994a, 1996a; Bottorff et al., 1995). Alignment of this domain with the A-helix of CAMP-dependent protein kinase suggests potential long range interactions with the activation lip (Herberg et al., 1997). This is supported by the synergistic enhancement of rates between Nterminal deletion and phosphorylation site mutations (Mansour et al., 1996a), as well as by deuterium exchange studies showing that both types of mutations enhance the flexibility within the N-terminal ATP-binding lobe (Resing and Ahn, 1997). Sequences in MKKl and MKK2 inserted between subdomains 9 and 10 of the conserved core are proline rich, containing consensus sequences for potential SH3 domain interactions. In MKK1, this domain is important for interactions between MKK and Raf-1 (Jelinek et al., 1994; Papin et al., 1996). Deletion of these residues in a constitutively active MKK background interferes with ERK activation and growth signaling in cells (Catling et al., 1996), suggesting that this domain is involved in substrate recognition in vivo, despite the fact that the deletion mutants still phosphorylate ERK in vitro. In

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MKKl, the insert contains four phosphorylatable residues that are targets for autophosphorylation ( Ser,,,, Tyr300)as well as heterologous phosphorylation by ERK1/2 and cyclidcdk2 (Thr2,6, Thr,,,) or PAKl (Ser298) (Brunet et al., 1994a; Saito et al., 1994; Mansour et al., 1994b; Rossomando et al., 1994; Resing et al., 1995; Frost et al., 1997). In MKK2, phosphorylatable residues are only found at Thr,,, and Ser,,,. Phosphorylation of these sites could potentially mediate MKURaf-1 interactions (Jelinek et al., 1994), feedback regulation of MKK activity by ERK (Brunet et al., 1994a), or cross-regulation of MKKl with PAK-dependent signaling pathways (Frost et al., 1997). In vitro, MKKl and MKK2 are targets for phosphorylation and activation by at least three protein kinase families. Raf-1 and B-Raf phosphorylate MKKl and MKK2, whereas A-Raf is a weak activator of MKK1/2 in intact cells and shows greater selectivity for MKKl in vitro (Wu, X. et al., 1996; Pritchard et al., 1995).MKKl and MKK2 are also activated by the germ cellspecific protein kinase, C-MOS,which controls meiotic cell division (Posada et al., 1993; Nebreda et al., 1993). Because MEKK and several isoforms are potent regulators of stress-activated protein kinase pathways, the discussion of MEKKs will be deferred to the next section. However, MEKKs and Tpl2 phosphorylate MKKl in vitro and under some conditions of expression in vivo (Yan and Templeton, 1994; Xu et al., 1995; Blank etal., 1996; Salmer6n et al., 1996). These enzymes phosphorylate both of the serine residues within the activation loop, although Mos preferentially phosphorylates Ser222, MEKKl and STEll prefer Ser218,and Raf-1 phosphorylates both sites equally well (Yan and Templeton, 1994; Alessi et al., 1994; Gotoh et al., 1994; Resing et al., 1995).In vitro, this activation is followed by intramolecular autophosphorylation at Thr23, Ser298,and Tyr300(Resing et al., 1995).

3. Raf Raf can be regulated by several mechanisms, the combination of which leads to full activation by growth regulatory signals. In addition to the Cterminal catalytic domain (amino acids 335-627), Raf-1, A-Raf, and B-Raf contain N-terminal regulatory domains within a conserved region 1 (CR1, amino acids 61-194 numbered as in Raf-1), which include a Ras- binding domain (RBD, amino acids 51-131) and a cysteine-rich domain containing a zinc finger motif (amino acids 139-184). A conserved region 2 (CR2, amino acids 254-269) and the very C terminus are important for binding the regulatory 14-3-3 protein. A basic model for Raf-1 regulation involves membrane recruitment, enabling its interactions with upstream regulators. Raf-1 recruitment is mediated by Ras-GTP (Marais et al., 1995), involving interactions between the Ras-binding and Cys-rich domains of Raf, respectively, with Switch I (amino

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acids 30-38) and Switch I1 (amino acids 60-76) effector regions of Ras-GTP (Moodie et al., 1993; Wojtek et al., 1993; Hu, C. et al., 1995; Brtva et al., 1995; Drugan et al., 1996). The GTP/GDP exchange of p21Ras is catalyzed by Ras guanine nucleotide exchange factors, including mammalian son-ofsevenless (Sos). Growth factor stimulation of GTP/GDP exchange entails recruitment of Grb2-Sos to membrane-bound Ras via direct interactions between receptor and Grb2 or indirect interactions through adaptor proteins such as Shc or IRS-1 (reviewed by Pawson, 1995). Disruption of either interaction leads to the suppression of transformation by v-ras, thus both interactions are important for Ras signaling (Fridman et al., 1994; Brtva et al., 1995). The importance of membrane recruitment was illustrated by the observation that membrane targeting of Raf-1 by C-terminal farnesylation or N-terminal myristylation transforms cells without participation of Ras (Leevers et al., 1994; Stokoe et al., 1994). A cocrystal structure of Raf-RBD and Rap complexed with a nonhydrolyzable GTP analog reveals close contacts between residues in the Raf-RBD and residues conserved between Ras and Rap (Nassar et al., 1995), suggesting how analogous interactions between Ras and Raf might be structured. Phosphorylation of Raf-1 appears to be a key regulatory mechanism. After purification from Sf9 cell expression, Raf-1 is phosphorylated at Ser,,, Ser,,,, Ser,21, Tyr340and Tyr341, and autophosphorylated at Thr,,, (Fabian et al., 1993a; Morrison et al., 1993). Raf-1 activation appears to require phosphorylation at one or both tyrosine residues because mutation of these sites blocks activation by Ras and because protein tyrosine phosphatases inactivate Raf-1 (Dent et al., 1995; Jelinek et al., 1996). Likely candidates for regulatory tyrosine kinases are Src and Fyn, based on studies demonstrating Raf-1 activation on coexpression with Src in a manner dependent on Tyr340/341(Fabian et al., 1993; Cleghon et al., 1994; Marais et al., 1995). Raf-1 activation by Src requires a functional RBD, suggesting that membrane recruitment is required for tyrosine phosphorylation (Marais et al., 1995). Ras-Raf interactions are not as important in Sf9 coexpression systems, presumably due to high expression levels (Fabian et al., 1994). Other candidates are the JAK tyrosine kinases, which appear to regulate Raf-1 in systems involving signaling through receptors for cytokines such as erythropoetin, interferon-y, and growth hormone (Miura et al., 1994; Winston and Hunter, 1995; Xia et al., 1996). JAK2 directly phosphorylates Raf-1 in vitro, although Tyr340/341account for only 75% of the incorporated label from 32P-ATP (Xia et al., 1996). Involvement of serinekhreonine kinases is indicated by reports of Raf-1 activation following its direct phosphorylation by protein kinase C (PKC) at Ser,,, (Sozeri et al., 1992; Kolch et al., 1993; Carroll and May, 1994)or by a ceramide-activated protein kinase at Thr,,, (Yao et al., 1995). Inhibition of Raf-1 on phosphorylation by CAMP- dependent protein kinase at Ser,, or Ser,,, has also been reported (Wu et al., 1993b;

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Mischak etal., 1996);phosphorylation at Ser,,, may be required for Raf activation through its interaction with 14-3-3 (Muslin et al., 1996). ERK also phosphorylates Raf-1, with no clear-cut effect in vitro, although it may suppress Raf-1 activity in viuo (Anderson et al., 1991; Ueki et al., 1994). In support of this, cellular stimulation of ERK leads to retardation of Raf gel mobility, which correlates with Ser621,624phosphorylation and lowered Raf activity (Ueki et al., 1994; Ferrier et al., 1997). Raf-1 heterocomplex formation has been proposed to be an important aspect of regulation. Physiologically, Raf-1 oligomerization may be accomplished through its interaction with 14-3-38, q, 5 and 0 (Fantl et al., 1994; Fu et al., 1994; Irie etal., 1994; Aitken et al., 1995; Papin et al., 1996),members of a large conserved acidic protein family that interact with many targets, including Raf-1, B-Raf, Bcr, polyomavirus middle T antigen, cdc25 phosphatases, phosphatidylinositol 3' kinase, Cbl, and protein kinase C-8 (reviewed by Aitken 1995; Meller et al., 1996). Residues within the C terminus of 14-3-3 interact with CR2 and the C terminus of Raf-1, recognizingphosphoserine at SerZs, and Ser,,, (Luo etal., 1995; Muslinetal., 1996). The importance of this interaction on Raf-1 regulation is indicated by several studies demonstrating activation of Raf-1 by 14-3-3 on coexpression in cells or in cell-free extracts (Freed et al., 1994; Fantl et al., 1994; Fu et al., 1994; Irie et al., 1994; Li et al., 1995). However, activation has not been observed in all cases (Michaud et al., 1995; Suen et al., 1995), and, in fact, 143-3 binding may stabilize an inactive form of Raf, based on Raf activation by disruption of 14-3-3 interactions. The ability of 14-3-3 to dimerize raises the possibility that 14-3-3 proteins may mediate the heterocomplex formation of Raf-l with other signaling molecules. For example, Raf-l association with Bcr has been demonstrated, mediated by their mutual interaction with 14-3-3 (Braselmann and McCormick, 1995). A 14-3-35 mutant that is unable to dimerize binds efficiently to Raf-1, but favors the inactive form of the kinase, suggesting that dimerization may be needed for Raf activation (Luo et al., 1995). Consistent with this, activation of basal Raf-1 and MKK1/2 activity was demonstrated on homodimerization of FKBP-Raf-Ucyclophilin or bacterial DNA gyrase-Raf-1 chimeras in response to bifunctional ligands FK1012A or coumermycin, respectively (Luo et al., 1996; Farrar et al., 1996). In vitro, Raf-1 elutes by gel filtration within protein complexes greater than 300 kDa containing several proteins with chaperone function, such as hsp90, Cdc37, and hsp56 (Wartmann and Davis, 1994; Stancato et al., 1994). Formation of hsp90 complexes appears to be important for Raf-1 signaling because the hsp90 ligand, geldanamycin, blocks growth factor signaling through the disruption of Raf-UMKK complexes (Schulte et al., 1996). However, no evidence exists for growth factor regulation of Raf-l:hspSO association or dissociation. Most likely, hsp90 interacts with Raf during folding, although hsp90 and 14-3-3 binding to Raf

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also protect against Raf-1 dephosphorylation and inactivation by PTPlB (Jelinek et af., 1996). Two additional interacting proteins may be important for Raf-1 activation. The kinase suppressor of Ras (KSR), a protein kinase closely related to Raf, was identified as a positive modifier of Ras signaling in Drosophila melanogaster and Caenorhabditis elegans (Kornfeld et al., 1995a; Sundaram and Han 1995; Therrien et af., 1995). Although direct regulation of Raf-1 by KSR has not been observed, biochemical experiments show association of human KSR with Raf, MKK1, and ERK and enhancement of signaling through the MKKERK pathway on coexpression with v-Ras in mammalian cells or Xenopus oocytes (Therrien et al., 1996). Direct stimulation of Raf1 by BAG-1, a protein that binds to and enhances the antiapoptotic effects of Bcl-2, has been demonstrated in vitro (H. H. Wang et al., 1996). Thus, BAG-1 may bind to and recruit Raf to Bcl-2. Involvement of Raf-1 in Bcl-2 regulation of apoptosis is suggested by biochemical interactions between Raf-1 and Bcl-2 and the observation that Bcl-2 and the proapoptotic Bc12 homolog, BAD, are both substrates for phosphorylation by Raf-1 (Wang et af., 1994; Blagosklonny et al., 1996; Zha et af., 1996). Other potential membrane activators of Raf-1 include lipids and lipid metabolites. In vitro, Raf-1 can be activated by direct phorbol ester binding, mediated through its zinc finger domains, which are homologous to those on several PKC isoforms (Luo et al., 1997). Raf-1 also binds to phosphatidylserine, phosphatidic acid, and ceramide (Ghosh et af., 1996; Huwiler et al., 1996). Binding of ceramide enhances Raf-1 activity in vitro and in vivo, and inhibition of phosphatidic acid synthesis in cells blocks Raf-1 membrane translocation. Finally, Raf-1 activation has been shown under certain conditions to be retarded by the inhibition of phosphatidylinositol 3' kinase, suggesting a possible role for lipid products of this enzyme in regulating Raf1 activity (Cross et af., 1994; J. Huang et al., 1995). In contrast to Raf-1, which is ubiquitous, other forms of Raf show more restricted tissue expression. A-Raf is mainly expressed in steroid responsive ( e g , urogenital) tissues (Winer and Wolgemuth, 1995; Lee et al., 1996).Several isoforms of B-Raf exist, many of which are selectively expressed in neuronal tissue (Barnier et af., 1996). Comparison of B-Raf to Raf-1 reveals similar but nonidentical mechanisms of regulation. Like Raf-1, B-Raf interacts with Ras-GTP and forms large molecular weight complexes that include hsp90 and 14-3-3 (Moodie et af., 1994; Jaiswal et al., 1996; Papin et al., 1996);however, unlike Raf-1, B-Raf complexed with 14-3-3 proteins retains its activity (Yamamori et al., 1995). B-Raf has a higher basal activity than Raf-1,ascribed to negatively charged Asp residues at positions equivalent to Tyr340and Tyr341 in Raf-1, and it is not as highly phosphorylated on tyrosine in response to factor stimulation, although it is still phosphorylated on Ser/Thr (Stephens et af., 1992; Jelinek et af., 1996). Moreover, B-Raf is stim-

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ulated by interaction with Rapl-GTP (Ohtsuka et al., 1996; Vossler et al., 1997), whereas RaplB-GTP binds but does not lead to Raf-1 activation (Zhang et a/., 1993). Dependence on phospholipid activators also differ; thus, phosphatidylserine stimulates B-Raf activity in a Ras-dependent manner, whereas phosphatidylinositol, phosphotidylserine, and phosphatidylethanolamine all inhibit RaplB-stimulated B-Raf activation (Kuroda et al., 1996). 4. Mos

The serinekhreonine protein kinase c-Mos regulates meiosis in vertebrate oocytes (Singh and Arlinghaus 1992; Yew et al., 1993). Its specific expression in germ cells is regulated by somatic cell repressor DNA-binding elements within the Mos promoter (Xu and Cooper, 1995), although somatic cell expression has also been reported (Leibovitch et al., 1993; Gao et al., 1996). Xenopus laevis oocytes stimulated to undergo meiotic cell division upregulate Mos protein levels by cytoplasmic 3’ polyadenylation and stabilization of message (Sheetset al., 1994,1995; Gebauer et al., 1994; StebbinsBoaz et al., 1996). Meiotic cell division, which can be induced by injection of Mos mRNA or protein into oocytes, requires activation of MKK and ERK; these are maximally active at metaphase I and I1 (Kosako et al., 1994a; Gotoh et al., 1995a; Roy et al., 1996). In vitro, Mos phosphorylates MKKl at regulatory Ser,,, and Ser,,, phosphorylation sites (Posada et al., 1993; Nebreda et al., 1993; Resing et al., 1995), and maturation through both meiosis I and I1 can be mimicked by the injection of constitutively active mutant MKKl or thiophosphorylated ERKl (Haccard et d., 1995; Gotoh et al., 1995a), thus MKK is a key target for Mos in this process. Regulation of Mos- or progesterone-dependent meiosis also involves Raf-1 activation, as indicated by the interference of germinal vesicle breakdown on the expression of inactive dominant negative Raf mutants (Muslin et al., 1993; Fabian et al., 1993); however, the ability of both Raf-1 and Mos to directly activate MKK suggests convergence of Raf and Mos signaling rather than sequential activation. Maturation of oocytes into meiosis I1 is inhibited by cycloheximide. This is due in part to a requirement for further synthesis of Mos protein, in a secondary induction that appears to require ERK activity (Yewet al., 1992; Gotoh et al., 1995a; Roy et al., 1996; Matten et al., 1996). Phosphorylation of Mos at Ser, enhances Mos protein levels (Nishizawa et al., 1992) and may lead to stable MKK interactions (Chen and Cooper, 1995). Ser, is phosphorylated by ERK in vitro, suggesting that in addition to message stabilization, Mos is regulated by positive feedback involving direct phosphorylation by ERK (Matten et al., 1996). Importantly, Mos functions as a cytostatic factor, elevating MPF activity and thus maintaining metaphase I1 arrest prior to fertilization. Following fer-

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tilization, calpain-mediated degradation of Mos is followed by a rapid decay of ERK activity, releasing cell cycle arrest. That ERK is a key component of cell cycle arrest is indicated by the cytostatic arrest of two cell embryos on microinjection of constitutively active MKK or thiophosphorylated ERK (Haccard et al., 1993; Kosako et al., 1994b). Targeted gene disruption of Mos in mice yields mouse oocytes that are unable to arrest in metaphase I1 and undergo parthenogenetic activation (Colledge et al., 1994; Hashimoto et al., 1994). Based on studies examining the effects of Mos on meiosis, ERK is implicated in metaphase arrest, meiotic spindle formation, and polar body degradation, and in the prevention of pronuclear envelope formation until fertilization (Verlhac et al., 1993; Moos et al., 1996; Choi et al., 1996 a&). MPF activation by Mos likely involves protection of cyclin B from proteolysis. Although some studies have shown suppression of cdc2kyclinB activation by dominant-interfering ERK mutants, the results have been mixed and a direct link with the MAPK pathway has not been established (Nebreda and Hunt, 1993; Pomerance et al., 1996; Huang and Ferrell 1996a). However, egg extracts induced to undergo spindle checkpoint arrest show high ERK activity and cyclin B levels. Arrest is released on inactivation of ERK by MAP kinase phosphatase-1, suggesting that ERK may also affect the mechanisms involved in cyclin degradation (Minshull et al., 1994).

5. SIGNALING THROUGH INTRACELLULAR CALCIUM MKK/ERK can be stimulated by elevation of intracellular calcium with calcium ionophores, thapsigargin, elevated extracellular calcium, or membrane depolarization (Chao et al., 1994; Rosen et al., 1994; S. Huang et al., 1995; Kurino et al., 1995; Bogoyevitch et al., 1996). In many cases, activation of Raf-1 and/or Ras in addition to MKKERK can be demonstrated, suggesting regulation of targets upstream of Ras. One candidate is Pyk2, a member of the focal adhesion kinase (FAK)family of nonreceptor protein tyrosine kinases, which is expressed in neuronal cells and tyrosine phosphorylated in response to calcium influx and membrane depolarization (Lev et al., 1995; Dikic et al., 1996).Forced expression of Pyk2 activates ERK, and dominant negative mutant Pyk2 blocks ERK activation by carbachol. Pyk2 also associates with Grb2 and Sos in intact cells following membrane depolarization or carbachol treatment of PC12 cells, suggesting a mechanism for its activation of Ras. An alternative (but potentially related) means of activating MKK/ERK by calcium involves Ca2+-calmodulin (CaM)-dependent protein kinases. Overexpression of Ca2+-CaM kinase IV in combination with its activator, CaM kinase kinase, led to a two- to fivefold activation of several MAPKs in PC12 cells, including JNWSAPK, p38, and ERK2 (Enslen et al., 1996). An involvement of Ca2+-CaMkinase I1 in the regulation of MKWERK is also sug-

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gested by the inhibition of norepinephrine activation of ERK by the Ca2+CaM kinase I1 inhibitor KN-93 in aortic smooth muscle cells (Muthalif et al., 1996). However, the involvement of calmodulin-regulated kinases in activating upstream regulators such as Raf-1 or Ras has not been established.

6. SIGNALING THROUGH HETEROTRIMERIC G PROTEINS Activation of MKWERK by heterotrimeric Gai, Gao, or Gaq proteincoupled receptors occurs in response to many agonists (e.g., lysophosphatidic acid, carbachol, endothelin, prostaglandin F,, bombesin, and thrombin). Overexpression of constitutively active GTPase-deficient Gai, Ga,, or G, mutants in various cell lines also leads to MKK/ERK activation (Gupta eta?., 1992; Faure et al., 1994; Pace et al., 1995). G,, subunits appear to be involved in the mechanism of Gai-coupled ERK regulation because activation can be suppressed by peptides corresponding to the C-terminal py-binding domain of the P-adrenergic receptor kinase (Koch et al., 1994) and because ERK is activated on expression of GP, subunits (Faure et al., 1994; Crespo et al., 1994; Ito et al., 1995; Hawes et al., 1995). G subunits appear to activate Ras, Raf, MKK, and ERK through a Pd?-independent mechanism that involves tyrosine phosphorylation of Shc and signaling to Ras through Shc-Grb2-Sos interactions (Van Biesen et al., 1995; Hawes et al., 1995). The tyrosine kinases Src and Syk are implicated in this process (Luttrell et al., 1996; Wan et al., 1996). In addition, the sensitivity of this pathway to wortmannin suggests a role of phosphatidylinositol 3' kinase downstream of G,, and upstream of Ras (Hawes et al., 1996). G-proteincoupled a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) activation of ERK resulted in association of G,, with a complex containing Ras, Raf, MEK, and ERK (Wang and Durkin, 1995), indicating that heterocomplex formation may occur between kinases and heterotrimeric G proteins. In contrast to Gai, stimulation of MKKERK through Gaq-or Gao-coupled receptors is independent of G, or Ras and instead activates Raf/MKK/ERK through a PKC-dependent meckanism (Van Biesen et al., 1996; Hawes et al., 1996). This may be a cell type-specific result because signaling through Gag in cardiac myocytes or CCL39 fibroblasts led to Ras/Raf activation through Shc-Grb2-Sos interactions in a PKC-independent manner (Y. Chen et al., 1996b; Sadoshima and Izumo, 1996). Candidate tyrosine kinase mediators of ERK activation through Gffqcoupled signaling are Lyn or Syk (Wan et al., 1996). Pyk2 appears to play an important role in heterotrimeric G protein signaling as well because ERK stimulation by agonists of Gai- or Gaq-linked pathways was suppressed by the dominant negative mutant Pyk2 in a process dependent on Src activation (Dikic et al., 1996). Under these conditions, Grb2 and Sos were required for ERK activation, indicating that signaling is Ras dependent.

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Gas-mediated signaling through CAMP-dependent protein kinase suppresses MKWERK activation in many systems (Wu et al., 1993b; Sevetson et al., 1993; Graves et al., 1993; Cook and McCormick, 1993; Hordijk et al., 1994), providing an explanation for the commonly observed antagonistic effects of cAMP on cell growth. One target for CAMP-dependent protein kinase inhibition is Raf-1, which is directly phosphorylated at Ser,, and Ser,,, in vitro (Wu et al., 1993b; Mischak et al., 1996).However, inhibition of ERK signaling is not observed in all cells and cAMP modulates the kinetics but not the magnitude of ERK activation under some conditions (McKenzie and PouyssCgur, 1996). In PC12 cells, agonists of cAMP signaling actually stimulate MKKERK (Frodin et al., 1994; Young et al., 1994; Vossler et al., 1997). This appears to result from the predominant role of B-Raf in regulating the MKKERK pathway in PC12 cells in that cAMP activates guanine nucleotide exchange of Rapl, which in turn activates B-Raf in a Rasindependent manner (Vossler et al., 1997).

7. SIGNALING THROUGH PROTEIN KINASE C A long-standing problem that is still unresolved is the mechanism by which PKC regulates MKK and ERK. Phorbol esters activate ERK in nearly every cell, and many reports exist of agonist stimulation of ERK that is at least partially blocked on PKC inhibition by extended phorbol ester treatment, low molecular weight inhibitors, or dominant negative PKC mutants (Hoshi et al., 1989; Ueda et al., 1996; Hundle et al., 1995; Berra et al., 1995). In lymphocytes, phorbol 12-myristate 13-acetate (PMA) leads to Ras-GTP elevation (Downward et al., 1990), and activation of ERK by PKC in broken cell extracts has been shown to require Ras (VanRenterghem et al., 1994).Therefore, one effect of PMA is to probably regulate Raf through the activation of Ras. The Pyk2 tyrosine kinase is a candidate for a Ras activator because it is also stimulated by phorbol ester (Lev et al., 1995).This may explain why PMA-dependent ERK activation is sensitive to tyrosine kinase inhibitors (Seger et al., 1995). However, Ras-independent activation of Raf-1 has also been observed (Ueda etal., 1996).An alternative mechanism to account for this involves direct phosphorylation of Raf by PKC-y, resulting in Raf-1 activation in vitro, as mentioned earlier (Sozeri et al., 1992; Kolch et al., 1993; Carroll and May, 1994). Interestingly, a Raf-1 chimera substituting the zinc finger domain with the analogous region from PKC is strongly responsive to phorbol ester in a manner that is unaffected by phorbol ester downregulation of PKC, suggesting that the zinc finger in Raf may be involved in the response to PMA (Luo et al., 1997). Although a direct comparison of the effects of individual PKC isoforms on MKWERK activation is still lacking, various reports have documented the involvement of PKC a,PI, y, 6 , E, and L in MKWERK activation (Ueda et al., 1996; Yamaguchi et al., 1995a; Hundle et al., 1995; Berra et al., 1995; Young et al., 1996).

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8. SIGNALING THROUGH JAWSTAT PATHWAYS Cytokine signaling pathways lead to interactions between receptors and members of the Janus family of tyrosine kinases (JAKs1, 2, and 3, TYK2), resulting in transphosphorylation and activation of kinase activity. STAT transcription factors are recruited to the receptor complex via SH2 domain interactions where they become tyrosine phosphorylated and dimerize through intermolecular interactions between N-terminal SH2 domains and C- terminal phosphotyrosine motifs (reviewed by Ihle, 1996). The resulting hetero- or homodimers translocate to nuclei where they mediate transcription by interaction with DNA response elements within cytokine-regulated enhancers, such as the interferon-? site (GAS) response element. The cytokine regulation of MKWERK in some cases may be explained by Janus kinase activation because growth hormone stimulation of ERK2 is blocked by dominant interfering mutants of JAK2 (Winston and Hunter, 1996). Expression of dominant negative mutant ERK2 blocks interferon-P-induced transcription, and treatment of cells with the MKKl inhibitor, PD98059, blocks IL-6-induced transcription, indicating a role for ERK in cytokine signaling through STAT1 or STAT3 (David et al., 1995; Bhat et af., 1996). A reasonable model would involve JAK-dependent activation of Ras through a Shc/Grb2-coupled pathway, as documented in several cases (VanderKuur etaf., 1995; He etal., 1995; Chauhan et al., 1995).However, not all cytokine receptor pathways utilize JAK for activating ERKs (Miura et af., 1994), indicating alternative mechanisms for carrying out these processes. Cross-regulation between JAK and ERK pathways also involves STAT regulation by ERK phosphorylation. Serine phosphorylation of STAT1 and STAT3 is necessary for full transcriptional activity, most likely stabilizing STAT dimers and STAT-DNA complex formation (Wen et af., 1995; X. Zhang et al., 1995). Phosphorylation of STAT1 or STAT3 at Ser,,, is important for this effect. These sites lie within the consensus sequence for ERK phosphorylation (Pro-X-Ser(P)-Pro), thus allowing positive feedback of STAT signaling by MKWERK. 9. SIGNALING THROUGH PHOSPHATIDYLINOSITOL 3’ KINASE

Evidence for an involvement of phosphatidylinositol3’ kinase (PI3’K) on MKKERK activation is based primarily on the suppression of signaling by pharmacological inhibitors or dominant negative mutants of PI3’K. Although PU’K signaling is likely to be more important in stress-activated kinase pathways, the PI3’K inhibitors wortmannin or Ly294002 (Powis et d., 1994; Vlahos et al., 1994) inhibit ERK activation in response to insulin, serum, vasopressin, platelet- activating factor, growth hormone, GPVsubunit expression, and T-cell receptor activation (Cross et af., 1994; Welsh et al.,

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1994; Ferby et al., 1994; Nishioka et al., 1995; Urich et al., 1995; Kilgour et al., 1996; Von Willebrand et al., 1996). Supporting evidence shows inhibition of ERK following the expression of dominant negative mutant PI3’K and ERK activation by constitutively active mutant PI3’K (Hu, Q. et al., 1995b; Von Willebrand et al., 1996). However, various studies have yielded conflicting results. In some cases, wortmannin or Ly294002 blocks ERK activation at doses uncorrelated with their inhibition of PI3’K, suggesting nonspecific mechanisms of action (Frevert and Kahn, 1997). Regulation of ERK by PI3’K might be mediated through Ras, which binds directly to the p l 1 0 catalytic subunit, resulting in elevation of PI3’K activity (Rodriguez-Viciana et al., 1996).

B. Stress-Activated Protein Kinase Pathways 1. STRESS-ACTIVATED MAPKs: JNWSAPK AND p38 MAPK

NH,-terminal cJun kinase (JNK)/stress-activated protein kinase (SAPK) and p38 MAP kinase are implicated in responses to cellular stress, inflammation, and apoptosis. Members of both families are ubiquitously expressed and are activated in response to lipopolysaccharides and proinflammatory cytokines interleukin-1 or tumor necrosis factor (TNF-a), ionizing or ultraviolet (UV) radiation, inhibitors of translation such as cycloheximide or anisomycin, cancer chemotherapeutics, tumor promoters, heat shock or hyperosmotic stress (DCrijard et al., 1994; Kyriakis et al., 1994; Han et al., 1994; Raingeaud et al., 1995; Y. Chen et al., 1996a; Kuroki et al., 1996; Osborn and Chambers, 1996).These are distinct from cell growth and transformation responses controlled by MKKERK, although the stress-activated kinases can be activated by growth stimuli under some conditions (Fig. 1). JNK/SAPK was first identified through purification as a cycloheximideactivated MAP2 kinase (Kyriakis and Avruch, 1990) as well as a protein kinase that copurified with and phosphorylated c-Jun (Adler et al., 1992; Hibi et al., 1993). Subsequent cloning efforts revealed three genes encoding JNWSAPK isoforms: JNK1, JNK2, and JNK3 (corresponding to SAPKa, p, and y) (DCrijard et al., 1994; Kyriakis et al., 1994). Alternative mRNA splicing at the C terminus results in further diversification of the JNK/SAPKs into as many as 10 isotypes, including four forms of JNK1, four forms of JNK2, and two forms of JNK3 with molecular masses ranging from 45 to 57 kDa (Gupta et ul., 1996). p38 MAPK was first purified as a protein phosphorylated in response to lipopolysaccaride (Han et al., 1994) and as a “reactivating kinase” able to phosphorylate and activate phosphatase-treated MAPKAP kinase-2 (Rouse et al., 1994). Both studies led to cloning of a protein kinase sharing 52% amino acid identity with the S . cerevisiue HOG1 protein kinase that responds

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to osmotic stress (Brewster et al., 1993; Han et al., 1994). The functional similarity of JNWSAPK and p38 MAPK was established by complementation of yeast hog1A strains (Han et al., 1994; Galcheva-Gargova et al., 1994). Four forms of p38 MAPK (a,p, y, and 6, also known respectively as RWSAPK2a, SAPK2b, ERK6ISAPK3, and SAPK4), have been identified (Jiang et al., 1996; Z. Li et al., 1996; Goedert et al., 1997). p38 MAPK a and p are inhibited by novel cytokine-suppressive anti-inflammatory drugs (CSAIDs) (Lee et al., 1994), are sometimes referred to as CSAID-binding proteins (CSBP1 and CSBP2). An alternatively spliced variant of p38 MAPK, called Mxi2, is a C-terminal truncation of p38 MAPKa, discovered by its interaction with Myc and its interacting partner, Max (Zervos et al., 1995). The overall sequence identity among JNWSAPK, p38 MAPK, and ERKs is 4 0 4 5 % . All three enzymes share a common mechanism for activation through phosphorylation of a Thr-X-Tyr motif found in the activation lip (DCrijard et al., 1994; Kyriakis et al., 1994; Gupta et al., 1996).JNWSAPK is activated by the phosphorylation of Thr-Pro-Tyr, whereas p38 MAPK is phosphorylated at a Thr-Gly-Tyr motif. The length of the activation lip differs among ERK, JNWSAPK, and p38 MAPK, which contain 25,21, and 1 9 amino acid residues, respectively, which are located between the Asp-Phe-Gly and Ala-Pro-Glu conserved sequences in subdomains 7 and 8. The sequences in the N-terminal half of the activation lip also differ among the three MAPKs. However, these differences do not account for the specific recognition by upstream protein kinases because substitution of the ERK2 activation lip by p38 MAPK or JNWSAPK sequences does not affect ERK2 phosphorylation by MKKl (Robinson et al., 1996a). Sequences immediately C-terminal to the P+ 1 recognition site are similar in all three MAPKs, consistent with their common recognition of Sermhr-Pro motifs. Crystallographic studies of the inactive form of p38 MAPKa show similarities in the overall domain structure, but significant differences in the folding of the activation lip compared to that of inactive ERK2 (Wilson et al., 1996; Z. Wang et al., 1997). Presumably, conformational changes induced by phosphorylation of p38 MAPK lead to an activation lip structure similar to that of active ERK2.

2. STRESS-ACTIVATED MAP KINASE KINASES 3, 4 , 6 , AND 7 Polymerase chain reaction (PCR) strategies were used to identify the upstream activators of JNWSAPK and p38 MAPK from mammalian libraries, using yeast PBS2 (Brewster et al., 1993), Xenopus XMEK2 (Yashar et al., 1993), or Drosophila hemipterous (Glise et al., 1995) as templates (Sanchez et al., 1994; DCrijard et al., 1995; Raingeaud et al., 1996). To date, four distinct MKKs have been identified (Fig. 1).MKK3 selectively phosphorylates p38 MAPK (DCrijard et al., 1995; Raingeaud et al., 1996). MKK6 is closely

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related to MKK3 (80% sequence identity) and also phosphorylates p38 MAPK, although its basal activity is approximately 300-fold higher than that of MKK3 (Raingeaud et al., 1996; Han et al., 1996; Moriguchi et al., 1996b; Stein et al., 1996).Both MKK3 and MKK6 have been found in two splice variant forms (Han et al., 1996; Moriguchi et al., 1996a).MKK4 and MKK7 (also called JNKKl/SEKl and JNKK2, respectively) are regulators of JNWSAPK, although MKK4 also phosphorylates p38 MAPK in vitro (Sgnchez et al., 1994; Dirijard et al., 1995; Lin et al., 1995; Tournier et al., 1997). MKKs 3, 4, 6, and 7 range from 35 to 45 kDa and lack the proline rich insertion between subdomains 9 and 10 that is found in MKKl and MKK2, although MKK4 retains a consensus site for ERK phosphorylation at its N terminus and is a substrate for phosphorylation by ERK (Dirijard et al., 1995). MKKs 3,4, 6, and 7 are responsive to inflammatory cytokines, ultraviolet or y irradiation, translation inhibitors, and osmotic stress, as expected from the behavior of JNWSAPK and p38 MAPKs. In some cells, MKK4 may be preferentially activated by translation inhibitors, whereas MKK3 and MKK6 may be preferentially activated by hyperosmotic shock, although this appears to vary substantially according to cell type (Moriguchi et al., 1995b, 1996a; Cuenda et al., 1996; Meier et al., 1996; Zanke et al., 1996a). The activation lip of these enzymes all contain serine residues at the position homologous to Ser,,, in hMKK1 and threonines at the position occupied by Ser,,,. Activation of these MKKs occurs through dual phosphorylation at these sites because mutagenesis at both positions is needed to obliterate phosphorylation by upstream kinases (Yan et al., 1994; Zanke et al., 1996a). MKK5 was cloned as a MKK homolog with 45% identity to MKK1, identified with two alternatively spliced forms of 50 and 40 kDa (English et al., 1995; Zhou et al., 1995). Yeast two hybrid screens identified an ERK homolog, ERK5, a 90-kDa protein identified in separate studies as big MAP kinase 1 (BMKl), that contains a Thr-Glu-Tyr motif within its activation lip (Zhou et al., 1995; Lee et al., 1995). Both ERK5 and MKK5 contain sequences outside their consensus kinase core, suggesting the presence of cytoskeletal-binding motifs, and MKKS is recovered in the particulate fraction of cell extracts. Despite the interaction between these enzymes, no evidence exists that ERK5 can serve as a substrate for MKKS or any other MKK. To date, ERK5 has no known function; however, it is activated by hydrogen peroxide or sorbitol in smooth muscle cells (Abe et al., 1996), suggesting its regulation by oxidant or osmotic stress.

3. STRESS-ACTIVATED MAP KINASE KINASE KINASES

a. MEKK MEK kinases (MEKKs) are mammalian homolog of yeast kinases STEl 1 and Byr2, first discovered through PCR strategies by Lange-Carter et al.

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(1993). At least nine mammalian MEKKs have been identified so far, including MEKKs 1, 2, 3, and 4, MAPKKKS, Tpl-2, TAK1, ASK1, and NIK. MEKK1, originally identified as a 5’-truncated mouse clone encoding a 73kDa polypeptide (Lange-Carter et al., 1993), was later identified in rat as a full-length 195-kDa polypeptide consisting of a C-terminal catalytic domain and several N-terminal domains (proline-, cysteine-rich and pleckstrin homology domains) likely to be involved in protein-protein association (Xu et al., 1996). MEKKl phosphorylates MKK1, MKK2, and MKK4 in vitro, although expression studies indicate that JNWSAPK pathways regulated through MKK4 are preferentially activated over ERK pathways in mammalian cells (Yan et al., 1994; Minden et al., 1994a; Xu et al., 1995; Zanke et al., 1996a). Interestingly, MEKKl is a poor activator of p38 MAPK in vivo, despite the recognition of p38 MAPK by MKK4 in vitro; thus it has been proposed that the formation of complexes through interaction domains of various protein kinases are important determinants of specificity (Zanke et al., 1996a). Expressed MEKKl can be activated by nerve growth factor or phorbol ester through Ras-coupled pathways in PC12 cells (Lange-Carter and Johnson, 1994), and endogeneous proteins immunocross-reactive with MEKK can be activated by a peptide chemoattractant in neutrophils (Avdi et al., 1996). Because MEKKl is active when expressed in mammalian cells, the mechanisms that govern its regulation are unclear. The ability of MEKKl to recognize its substrates for phosphorylation might involve cellular targeting, consistent with the observation that the full-length enzyme is enriched within membrane fractions of cell extracts (Xu et al., 1996). MEKK2 and MEKK3 predict 70- and 71-kDa polypeptides, respectively, with 94% sequence identity to each other within their kinase domains (vs 50% identity to MEKK1) and greater divergence in their N-terminal domains (Blank et al., 1996). In vitro, MEKK2 phosphorylates MKKs 1 and 4, while MEKK3 phosphorylates MKKs 1,3, and 4 (Ellinger-Ziegelbauer et al., 1997; Blank et al., 1996; Deacon and Blank, 1997), although on expression in mammalian cells, both stimulate JNWSAPK and ERK, but not p38 MAPK. MEKK4 and MAPKKK5 are respectively 180 and 150 kDa polypeptides with 55% and 40% identity to MEKKs 1,2, or 3, and stimulate JNW SAPK, but not ERK or p38 MAPK following cell expression (X. Wang et al., 1996; Gerwins et al., 1997). Tpl-2 is an oncogene discovered as a locus of provirus insertion in Moloney murine leukemia virus-induced rat T-cell lymphomas (Patriotis et al., 1993,1994) and is >90% identical to the human and mouse Cot oncogene (Miyoshi et al., 1991). Tpl-2 encodes a 51- kDa polypeptide with 32% sequence identity to MEKKl within its kinase domain and is activated by deletion of 43 amino acids at its C-terminal tail (Patriotis et al., 1994). In vitro, Tpl-2 phosphorylates and activates MKKl and MKK4 (Salmer6n et

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al., 1996) and activates ERK and JNWSAPK on expression in mammalian cells. A protein kinase of 64 kDa with 30% sequence identity to the catalytic domain of MEKKl was revealed in screens for mammalian regulators of yeast STE7. This enzyme was named TGF-@-activatedkinase (TAKl) because it is rapidly activated and transcriptionally induced in response to TGF-@and bone morphogenic protein 4 (BMP4) and mediates transcriptional activation of TGF-@-responsiveenhancers (Yamaguchi et al., 1995b). In vitro, TAKl phosphorylates MKK4 and MKK6 but not MKK1, raising the possibility that it mediates TGF-P signaling through JNWSAPK pathways (Yamaguchi et al., 1995b; Moriguchi et al., 1996b). Two-hybrid yeast screens identified TAB, a novel protein that interacts with and enhances kinase activity of TAKl in intact cells (Shibuya et al., 1996).Thus, stress activated kinase cascades are implicated in transcriptional responses to TGF-p signaling. Apoptosis signal-regulating kinase (ASK1) has 30% sequence similarity to MEKKl and STEll in its kinase domain (Ichijo et al., 1997) and is more closely related to MEKKs SSK2 and SSK22 in the yeast HOG1 pathway (Maeda et al., 1995; Section VI,A,3). ASKl activates MKK3 and MKK4, but not MKKl in vitro, and activates the JNWSAPK and p38 MAPK pathways, but not ERK, following mammalian cell expression. Endogeneous ASKl is activated by TNF-a in several cell lines and its overexpression leads to apoptosis (Ichijo et al., 1997). Another MEKK homolog implicated in the cell death response is NF-&-inducing kinase (NIK), which was identified through its association with TNF receptor-associated factor 2 (TRAF2) adaptor (Malinin et al., 1997). Catalytically inactive NIK mutants block TNF-a or interleukin-1 signaling to NF-KB,and the expression of wild-type NIK inhibits cytotoxic responses to TNFa, suggesting a role for this enzyme in NF-KB-mediated resistance to cell death.

b. Mixed Lineage Kinases A second class of enzymes, distinct from MEKKs, also activate JNWSAPK and p38 MAPK pathways. Mixed lineage kinases (MLKs) contain leucine zipper domains for protein-protein interactions outside the conserved kinase core. Members of this family include MLK-2 (Dorow et al., 1993; Katoh et al., 1995; Hirai et al., 1997), MLK-3/SPRWPTK-l (Ezoe et al., 1994; Gallo et al., 1994; Ing et al., 1994) and DLWZPWMUK (Holzman et al., 1994; Reddy and Pleasure, 1995; Hirai et al., 1996). MLKl (Dorow et al., 1993) is a related enzyme that is not as thoroughly characterized (Fig. 1). MLK2 and MLK3/SPRK are 105- and 93-kDa polypeptides, each with a catalytic domain flanked by an SH3 domain on its N-terminal side and by two leucine zipper domains on its C-terminal side. Between the leucine zippers and a proline/glycine-rich C-terminal tail lies a Cdc42Rac-interactive

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binding (CRIB) motif (Ile-Ser-X-Pro-X,-,His-X-X-His)that enables interaction of these enzymes with Racl-GTP or Cdc42-GTP (Burbelo et al., 1995; Teramoto et al., 1996b). In vitro, MLK-2 and MLK3/SPRK phosphorylate and activate MKK4. Cell expression of either enzyme triggers both JNWSAPK and p38 MAPK, and coimmunoprecipitation of MLK3/SPRK with MKK4 or MKK6 has been observed (Rana et al., 1996; Tibbles et al., 1996), indicating a specific role for these enzymes in stress signaling pathways. DLWMUK is a 100-kDa polypeptide that migrates as a 130-kDa polypeptide on SDS-PAGE. It contains a catalytic domain flanked on its C-terminal side by a dual leucine zipper motif and proline/glycine rich regions at both N and C termini, although only an incomplete CRIB domain is present (Fan et al., 1996). DLWMUK also activates JNWSAPK and p38 MAPK when expressed in mammalian cells, and catalytically inactive DLWMUK can inhibit Ras-dependent JNWSAPK activation (Hirai et al., 1996; Fan et al., 1996). Furthermore, the activation of JNWSAPK is blocked by the expression of a catalytically inactive MEKKl mutant, indicating that DLK/MUK may directly or indirectly regulate MEKKl activity (Fan et al., 1996). Neither MLK3/SPRK nor DLWMUK show significant signaling through the MKW ERK pathway, although MLK-2 activates ERK to an extent comparable to p3 8 MAPK. However, detailed comparisons of the substrate specificities of these enzymes in vitro are still awaited. 4. UPSTREAM REGULATORS OF JNWSAPK AND p38 MAPK PATHWAYS

a . Rac, Rho, and Cdc42-Regulated Kinases In analogy to Ras-GTP regulation of MKWERK, members of the Rho family of small GTPases, including RhoA, Racl , Rac2, and Cdc42, are positive regulators of JNWSAPK and p38 MAPK pathways (Coso et al., 1995; Minden et d., 1995)(Fig. 1).These GTPases are implicated in regulating cytoskeletal reorganization. Rac and Cdc42 regulate cell motility through the formation of lamellipodia and filopodia at the membrane leading edge, whereas Rho is involved in the formation and maintenance of actin stress fibers and focal adhesion plaques as well as the formation of the actin contractile ring at cell division (Nobes and Hall, 1995). Mechanisms linking these proteins to the activation of various MKKs are still unclear but may involve the action of several protein kinases that couple Rac and Cdc42 to stress-activated kinase pathways. p21Ras-activated kinases (PAKs) are 60- to 70-kDa enzymes that are closely related to yeast STE20 (Leberer et af., 1992) within their C-terminal kinase domains; they interact with GTP-Rac and GTP-Cdc42, but not RhoA, through N-terminal CRIB domains. Four mammalian enzymes have been de-

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scribed so far, including PAK1, PAK2, PAK3/@PAK,and yPAK (Manser et al., 1994,1995; Martin et al., 1995; Bagrodia et al., 1995a; Teo et al., 1995). PAK activity and autophosphorylation are stimulated by GTP-Rac or GTPCdc42 in vitro and in vivo, whereas the expression of PAK in mammalian cells leads to activation of JNWSAPK and p38 MAPK (Bagrodia et al., 1995b; Zhang, S. et al., 1995; Frost et al., 1996). ERK is not a major target for activation by Rac or PAK; however, a potential mechanism for crossregulation is indicated by the ability of PAK to synergize with Raf-1 in stimulating MKK and ERK activity (Frost et al., 1996). This may be regulated by the phosphorylation of Ser,,, on MKKl by PAK because mutagenesis of this residue to Ala significantly reduces the synergy (Frost et al., 1997). The mechanisms by which PAK activates JNK are not clearly defined, but may involve signaling through MEKKs or MLKs. Dominant negative MLK3 has been shown to inhibit Cdc42 and Rac signaling to JNWSAPKs (Teramoto et al., 1996b). Alternatively, PAK3 interacts with phospholipase Cy, and PAKs 1 and 3 interact with the adapter protein, Nck, through SH3 domain interactions with N-terminal proline-rich motifs in PAK (Bagrodia, 1996a; Galisteo et al., 1996; Bokoch et al., 1996). PAKl binding to Nck is enhanced by PDGF, leading to elevated Nck phosphorylation, whereas PAKl activity is enhanced in response to EGF on coexpression with Nck. Thus, Nck might mediate signaling upstream or downstream of PAK. Although RhoA also regulates JNWSAPK and p38 MAPK pathways, this does not appear to involve PAK (Teramoto et al., 1996a). Instead, RhoA interacts with protein kinase N (PKN),a 120-kDa protein kinase C-related enzyme with N-terminal leucine zipper motifs (Mukai and Ono, 1994; Amano et al., 1996; Watanabe et al., 1996). Rho-PKN interactions lead to the activation of PKN in vitro and in vivo (Watanabe et al., 1996; Amano et al., 1996), and PKN translocates from cytosolic to nuclear pools following cell stress (Mukai et al., 1996), suggesting that this kinase mediates Rho-dependent activation of JNWSAPK or p38 MAPK. Rac, Rho, and Cdc42 also interact with RhoA-binding kinases a and @ (ROKs), 150- to 160-kDa protein kinases with lengthy coiled coil domains, related to human myotonic dystrophy kinase (Leung et al., 1995,1996; Ishizaki et al., 1996). These induce actin polymerization on cell expression (Leung et al., 1996) and are thus implicated in Rho-dependent cytoskeletal rearrangements. However, experiments introducing point mutations into Rac, Cdc42, and Rho effector domains have shown that the effects of these GTPases on PAK, JNWSAPK, and p38 MAPK activation are not correlated with their effects on ROK activation, lamellipodia and filopodia extension, or actin stress fiber formation (Lamarche et al., 1996; Joneson et al., 1996). This suggests that stress-activated kinase pathways are uncoupled from cytoskeletal reorganization events occurring in response to Rac, Cdc42, or Rho.

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b. Germinal Center Kinases Other protein kinases implicated in stress-activated signaling pathways are members of the germinal center kinase (GCK) family, including GCWRab8ip, HPK1, MST1, MST2, and SOKl (Katz et al., 1994; Creasy and Chernoff, 1995a,b; Ren et al., 1996; Hu, M. et al., 1996; Pombo et al., 1996), which are mammalian homologs of yeast STE20 and Spsl (Friesen et al., 1994). These enzymes are characterized by N-terminal catalytic domains followed by proline-rich regions, leucine-rich regions, and potential PEST sequences (but no Rac interaction motifs). Although none have been found to directly phosphorylate MKKs, on mammalian cell expression these enzymes activate stress-activated kinases, indicating an indirect regulatory role. The 97-kDa GCK is expressed in several tissues, but in lymphoid follicles is restricted to germinal centers, suggesting a role in B-cell development (Katz et al., 1994). It is stimulated in response to TNF-a, and its expression leads to MKK4 and JNWSAPK activation, but not ERK or p38 MAPK activation (Pombo et al., 1995). A closely related mouse homolog, Rab8ip, is found to coimmunoprecipitate and colocalize with Rab8, implicating this enzyme in Golgi vesicle targeting or fusion (Ran et al., 1996)(Fig.1). Hematopoetic progenitor kinase 1 (HPK1) is a 97-kDa enzyme that is found predominantly in blood cells and, when overexpressed, selectively activates JNK/SAPK (Kiefer et al., 1996; Hu, M. et al., 1996). The activation of JNWSAPK by HPKl is inhibited by dominant negative mutants of MEKK1, MLK-3, or MKK4. HPKl coprecipitates with and phosphorylates both MEKKl and MLK-3, suggesting a mode of regulation involving multicomponent complex formation. Mammalian STE20-like kinases (MSTl, MST2) and STE20/oxidant stress response kinase (SOK-1)also belong to the GCK family (Creasy and Chernoff, 1995a,b; Pombo et al., 1996). SOKl is activated by cytokines and oxidants, but does not appear to regulate JNK/ SAPK or p38 MAPK.

5. RECEPTORS AND SECOND MESSENGERS INVOLVED IN CELL STRESS Although JNK/SAPK and p38 MAPK pathways are stimulated by many different types of physical stressors, little is known about the mechanisms for sensing these stimuli. In yeast, HOG1 activation by hyperosmolarity involves at least two osmoreceptors, SLNl and SHOl (Maeda etal., 1995; see Section VI,A,3) which may be activated through mechanical membrane distortions. Physical membrane events are also possible in JNWSAPK activation, based on findings that UV light or hyperosmolarity induces clustering of EGF, TNF-a, or interleukin-1 receptors that might then signal in response to dimerization (Rosette and Karin, 1996). Alternative models invoke DNA damage as common signals induced by UV or y irradiation, chemical muta-

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gens, and cancer chemotherapeutic agents (e.g., cisplatinum, adriamycin, vinblastine, etoposide). The degree of JNWSAPK activation has been correlated with the number of DNA lesions induced by UV-B or W - C irradiation (Adler et al., 1995), although conflicting results have been obtained regarding the ability of UV to induce JNWSAPK activation in enucleated cells (Devary et al., 1993; Adler et al., 1995). Reactive oxygen intermediates (ROIs)such as hydroxyl-free radicals are generated by many agents that also activate JNWSAPK and p3 8 MAPK, including GTPase-deficient mutant Rac(Vall2) (Sulciner et al., 1996), suggesting that ROIs provide common signals to JNWSAPK pathways in response to irradiation, anticancer drugs, or cytotoxic cytokines. This model is supported by the potent activation of JNWSAPK by oxidants (e.g., H,O,) (Lo et al., 1996) and by the inhibitory effect of radical scavengers (e.g., N-acetyl-L-cysteine) on JNWSAPK activation by cell stress (Tao et a/., 1996). However, several studies on the effects of oxidants and antioxidants on JNK/SAPK are also conflicting (Yu, R. et al., 1996; Gomez del Arc0 et al., 1996), and so far a causal role of ROIs in signaling through the kinase pathways has not been definitively established. Ligand-receptor-mediated pathways implicated in JNWSAPK and p38 MAPK activation include those involved in inflammatory and/or apoptotic responses, triggered by receptors for TNF-a, IL-1, and Fas ligand as well as the CD14 receptor for lipopolysaccharide. Rac or Cdc42 are activated in response to all of these stimuli and may be common signaling intermediates, although the mechanisms regulating their respective guanine nucleotide exchange factors are unknown. Receptor-mediated signaling may also involve several heterotrimeric Ga proteins that have been shown to stimulate JNWSAPK preferentially over ERK. Activation of JNWSAPK by GTPasedeficient mutant GmlZor GmI3occurs through mechanisms dependent on Rac and Cdc42 and can be blocked by dominant negative mutant MEKKl (Prasad et al., 1995; Collins et al., 1996; Voyno-Yasenetskaya et al., 1996). In addition, GP./ subunits mediate JNWSAPK signaling through Gmi-coupled muscarinic receptors (Coso et al., 1996). Ras is implicated in some of these studies, based on inhibitory effects of dominant negative mutant Ras (Prasad et al., 1995; Adler et al., 1996); however, in the majority of cases, stress-activated pathways are Ras independent. Abelson tyrosine kinase (Abl) is implicated in regulating cell growth, but is also activated by compounds that induce DNA damage, including ionizing radiation (Kharbanda et al., 1995a,b). Both v-Abl and Bcr-Abl activate JNWSAPK (Renshaw et al., 1996; Raitano et al., 1995).Furthermore, Able’cells generated by homologous recombination are unable to activate JNWSAPK in response to cisplatinum or l-P-D-arabinofuranosylcytosine (AraC), although cells still respond to W light and alkylating agents, implying the existence of convergent stress-signaling pathways (Kharbanda et al., 1995b; Pandey et al., 1996). These findings indicate an upstream regu-

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latory role for c-Abl, most likely mediated through Rac, because dominant negative mutant Rac inhibits v-Abl-induced JNWSAPK activation (Renshaw et al., 1996). Another tyrosine kinase implicated in JNWSAPK activation is Pyk2, which is activated in response to TNF-a, UV irradiation, and hyperosmolarity, and activates JNWSAPK on overexpression (Tokiwa et al., 1996). A calcium-dependent tyrosine kinase, partially purified and found to be related to Pyk2, was stimulated by thapsigargin and angiotensin I1 under conditions resulting in substantial JNK activation and minimal ERK activation (Yu, H. et al., 1996). Lipid second messengers are implicated in stress-activated kinase signaling. In response to TNF-a, interleukin-1, Fas ligand, or ionizing radiation, ceramide levels increase as a result of elevated sphingomyelinase activity. Activation of JNWSAPK is also observed on direct treatment of cells with cellpermeable ceramide analogs (Westwick et al., 1995; Pyne et al., 1996; Welsh, 1996; Coroneos et al., 1996). Lipid products of PI3’K may also be involved in signaling to JNWSAPK kinases. Elevation of Rac-GTP and activation of JNWSAPK are inhibited by wortmannin under some conditions (Hawkins et al., 1995; Ishizuka et al., 1996), and a constitutively active mutant of PI3’K stimulates JNWSAPK (Klippel et al., 1996). Thus, in addition to other Rac or Rho-like morphological responses (Reif et al., 1996), JNK/SAPK may be regulated by products of PI3’K through activation of Rac. Further evidence for a role of PI3’K in JNWSAPK signaling is provided by studies in cells derived from patients with ataxia telangiectasia (A-T), a deficiency in a PI3’K-like enzyme, where ionizing radiation (but not UV or anisomycin treatment) failed to activate JNWSAPK (Shafman et al., 1995). However, ionizing radiation induces an association between JNWSAPK and the p85 . subunit of PI3’K, mediated through Grb2 (Kharbanda et al., 1 9 9 5 ~ )Under the latter conditions, wortmannin stimulated JNWSAPK activity, suggesting inhibitory effects of PI3’K on JNK/SAPK activation. 6 . JNWSAPK AND p38 MAPK PATHWAYS IN APOPTOSIS

Many agents that activate JNWSAPK are cytotoxic, suggesting that this kinase pathway positively regulates cell death. In blood cells, cytokine-stimulated apoptosis is mediated through ceramide synthesis and can be induced by ceramide uptake into cells and antagonized by sphingosine 1-phosphate (Obeid et al., 1993; Cuvillier et al., 1996). A causal role for JNWSAPK in cell death is suggested by the finding that ceramide-induced apoptosis in U937 cells and UVC or y radiation-induced apoptosis in T cells can be inhibited by the expression of dominant negative mutant MKK4 (Verjeijet al., 1996; Chen, Y. et al., 1996a). Furthermore, ERK is activated by sphingosine 1-phosphate and the cytoprotective effect of this metabolite requires ERK

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activity because it is suppressed by PD98059 (Cuvillier et al., 1996; Pyne et al., 1996). Similar results have been reported in model systems for neuronal cell apoptosis. PC12 cells differentiated with nerve growth factor (NGF)undergo apoptosis following NGF withdrawal. Cell death is accompanied by the activation of JNWSAPK and p38 MAPK and appears to be dependent on these pathways, because expression of dominant negative mutant MKK3 or MKK4 promotes cell survival (Xia et al., 1995). PC12 cell survival is also enhanced by the expression of constitutively active MKKl (Xia et al., 1995). Thus, in neuronal and blood cells, relative levels of signaling through ERK vs JNWSAPK or p38 MAPK appear to regulate the choice between cell survival vs death. Consistent with this model, a role of JNWSAPK in epithelial cell apoptosis induced by integrin-extracellular matrix disruption (anoikis) is blocked by dominant negative mutant MKK4 (Frisch et al., 1996). Although JNWSAPK activation is correlated with apoptosis induced by ceramides and UV and y irradiation, the same may not be true for TNF-aor Fas-induced apoptosis. Thus, JNWSAPK activation was inhibited by dominant negative MEKKl or MKK4 in response to TNF-a or Fas, respectively, with no effect on ligand-induced cell death (Liu et al., 1996; Lenczowski et d., 1997). Correspondingly, apoptosis induced by TNF-a or Fas was inhibited by mutagenesis of the FasKNFR1 receptor interacting proteins, RIP or FADD, or with interleukin-1 converting enzyme (ICE) protease inhibitors, with no reduction in JNK activation. Although the p38 MAPK inhibitor, SB203580, had no effect on TNF-a- or Fas-mediated apoptosis (Juo et al., 1996; Bayaert et al., 1996),expression of active MKK3 or MKK6 augmented the Fas-dependent cell death response (Juo et al., 1996; Huang et al., 1997), indicating a potential involvement of MKK3/6 through a p38 MAPK-independent mechanism. Furthermore, whereas JNWSAPK and p3 8 MAPK are involved in some but not all apoptotic responses, activation of JNWSAPK during anoikis and Fas-dependent activation of p38MAPK in T cells can be inhibited by the CrmA protease inhibitor (Frisch et al., 1996; Juo et al., 1996), providing evidence for a positive feedback between stressactivated kinases and ICE proteases.

111. REGULATION OF MAPK PATHWAYS BY PROTEIN PHOSPHATASES Protein phosphatases are catagorized into two general groups, the protein phosphatases (PPs) and the protein tyrosine phosphatases (PTPs). Attenuation of signaling in the various MAPK pathways is controlled by members of both these families (Table I). The PPs specifically hydrolyze serinehhreo-

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Table I Regulatory Phosphatases in MAPK Pathways Phosphatasen Mammalian PPS PP2A

Target

Localization

Reference

ERK and MKK

Cytosol (mainly)

Anderson et al. (1990), Gomez and Cohen (1991), Sontag et al. (1993), Alessi et al. (1995), Cohen (1989)

ERK, JNK, p38

Nucleus

PAC 1

ERK and p38

Nucleus

MKP-2 (hVH-2, TYP- 1)

ERK, JNK, p38

Nucleus

MKP-3 (RVH-6, PYST1)

ERK

Cytosol

hVH-3 (B23)

ERK

Nucleus

M316

JNK and p38

Cell type dependent

hVH-5 VHR

ERK ERK

ERK, JNK, p38

Sun et al. (1993),Alessi et al. (1993), Noguchi etal. (1993),Zheng and Guan (1993),Brondello et al. (1995), Chu et al. (1996) Rohan etal. (1993), Ward et al. (1994), Chu et al. (1996) Guan and Butch (1995), Misra-Press et al. (1995), King et al. (1995), Chu et al. (1996),Brondello et al. (1997) Mourey et al. (1996), Muda etal. (1996b), Groom et al. (1996), Muda et al. (1996a) Kwak and Dixon (1995), Ishibashi et al. (1994) Theodosiou et al. (1996), Muda et al. (1996a) Martell et al. (1995) Ishibashi et al. (1992), Ishibashi et al. (1994) Groom et al. (1996 j, Muda et al. (1996b) Muda et al. (1997)

DSPs MKP-1 (CL100, ERP,3CH134, hVH-1)

MKP-X (PYST2) MKP-4 Saccharomyces cerevisiae

PPS PTC 1,2 PTPs PTP2,3

(HOG1 and PBS2)?

Maeda et al. (1994)

HOG1

Maeda et al. (1994),Jacoby et al. (1997),WurglerMurphy et al. (1997)

DSPs MSG5

FUS3 and MPKl

Doi et al. (1994), Watanabe et al. (1995) (continues)

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Table I (continued) ~

Phosphatasea

Target

Localization

Reference

Schizosaccharomyces pombe PPS Ptc1,2,3

(Spcl and Wisl)?

Shionaki and Russell (1995b)

PTPs PYPlJ-

spc 1

Shiozaki and Russell (1995a), Miller et al. (1995), Degols et al. (1996)

DSPs Dspl

Pmkl ~

Toda etal. (1996) ~

~~~~

aPPs, Ser/Thr protein phosphatases; PTPs, protein tyrosine phospharases; DSPs, dual specificity phosphatases.

nine phosphoesters and include PP1, PP2A, PP2B, and PP2C (Wera and Hemmings, 1995). Hydrolysis of tyrosine phosphoesters is catalyzed by the PTPs, which include a subfamily of dual specificity phosphatases (DSPs) capable of hydrolyzing both phosphotyrosine and phosphoserine/threonine (Fauman and Saper, 1996). Although PPs and PTPs have similar functions, their catalytic mechanisms and structures are different (reviewed by Denu et al., 1996a; Barford, 1996). The crystal structures of PP1 (Goldberg et al., 1995; Egloff et al., 1995), PP2B (Griffith et al., 1995; Kissinger et al., 1995), and PP2C (Das et al., 1997) have been determined, revealing a central P-cx-P-a-P motif containing a dinuclear metal ion center at the active site. Based upon structural data, a single-step catalytic mechanism has been proposed in which metalactivated water directly attacks the phosphorous center of the substrate. In contrast, PTPs do not require metal ions for catalysis, but utilize a catalytic cysteine present in the conserved active site sequence motif: His-Cys-X-X-Gly-X-X-Arg-Ser/Thr.Catalysis occurs in a two-step mechanism in which a phosphoenzyme intermediate forms at the cysteine followed by hydrolysis by water to restore the enzyme (Denu and Dixon, 1995a, 1996b). Comparison of the PTPlB structure (Jia et al., 1995) with that of the dual specificity phosphatase VHR (Yuvaniyama et al., 1996) suggests that the depth of the active site pocket determines substrate specificity. The shallow active site pocket of VHR accommodates phosphorylated serine, threonine, and tyrosine, whereas the deeper active site of PTPl B favors the longer phosphotyrosine side chain (Yuvaniyama et al., 1996).

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A. Dual Specificity Phosphatases Dual specificity phosphatases constitute the largest family of phosphatases capable of regulating MAPKs in mammalian systems. DSPs that modulate MAPK pathways are homologous, especially in their catalytic domains; each is related to the prototypical VH1 dual specificity phosphatase of vaccinia virus (Guan et al., 1991). This group of MAPK phosphatases (MKPs) includes the following members: MKP-1 (also named CL100,3CH134, hVH1, or ERP), MKP-2 (hVH-2 or TYP-l), MKP-3 (rVH-6 or PYSTl), MKP-4, MKP-X (PYST2), PAC1, hVH-3 (B23), M3/6, hVH-5, and VHR (Table I). Although similar in function, members of this family vary in substrate specificity, localization, regulation of gene expression, and tissue distribution. Because MAPKs require both threonine and tyrosine phosphorylation for full activity, dual specificity MKPs are in a unique position to regulate MAPK signal transduction cascades. MKP-1, the first of the MKPs to be characterized, inactivates ERK2 in vitro by concomitant dephosphorylation of phosphothreonine and phosphotyrosine residues (Charles et al., 1993; Alessi et al., 1993; Zheng and Guan, 1993b).ERKs are also effectively inactivated by MKP-1 in vivo (Sun et al., 1993; Duff et al., 1995). Similarly, PAC1, MKP2, and MKP-3 inactivate ERK1/2 both in vitro and in vivo, whereas hVH-3, hVH-5, and VHR do so only in vitro (Table I). The stress-activated kinases JNWSAPK and p38 MAPK are also substrates for MKP-1 and MKP-2, whereas PAC1 recognizes only p38 MAPK (Chu, Y., et al., 1996; Brondello et al., 1997). However, JNWSAPK inactivation requires neither MKP-1 nor MKP-2 in vivo, and ERK is a better MKP-1 substrate than JNWSAPK (Sun et al., 1994; Brondello et al., 1997). MKP-3 is specific for ERKs1 and 2, whereas M3/6 selectively inactivates JNWSAPK and p3 8 MAPK (Muda et al., 1996a; Groom et al., 1996). The substrate specificities of individual MKPs imply their differential ability to regulate various MAPK pathways. Indeed, MKP-3 blocks growth factor stimulation of ERKl in vivo, but not stress-induced activation of JNKUSAPK or p38 MAPK (Groom et al., 1996). Conversely, M3/6 is restricted to JNKUSAPK and p38 MAPK and has no effect on EGF stimulation of ERKl (Muda et al., 1996a).Overexpressed MKP1 is able to block both signaling pathways (Sun et al., 1993; Liu, 1995). In general, MKPs localize within nuclei, providing a mechanism for the inactivation of nuclear MAPK (Table I). MKP-1 is exclusively nuclear in both quiescent and stimulated cells (Brondello et al., 1995; Lewis et al., 1995), as are PAC1, MKP-2, and hVH-3 (Rohan et al., 1993; Guan and Butch, 1995; King et al., 1995; Kwak and Dixon, 1995). In contrast, microinjected M3/6 shows localization dependent on cell type, being nuclear in Swiss 3T3 cells, but cytoplasmic in MDCK and PC12 cells (Theodosiou et al., 1996).MKP3 and MPK-4 are excluded from the nucleus and thus appear to be a regulator of cytoplasmic ERKl and ERK2 (Groom et al., 1996; Mourey et al.,

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1996; Muda et al., 1996b, 1997).Cytoplasmic vs nuclear regulation of MAPK signaling functionally distinguishes MKP-3 and -4 from the other MKPs. Most MKPs are transcriptionally upregulated in response to mitogenic and stress stimuli, although in different ways. MKP-1 was originally identified as an immediate early gene responsive to oxidative stress and heat shock, as well as serum stimulation (Lau and Nathans, 1985; Keyse and Emslie, 1992). UV light and anisomycin treatment also rapidly enhance MKP-1 mRNA levels (Liu et al., 1995; Bokemeyer et al., 1996). PAC1 is a mitogen-induced early response gene that is predominantly expressed in hematopoietic tissues (Rohan et al., 1993). Similarly, induction of hVH-5 by NGF and insulin is characteristic of an immediate early gene (Martell et al., 1995). In contrast, the other MKPs differ from MKP-1 in their magnitude and kinetics of mRNA expression. hVH-3 is induced neither by oxidative stress nor UV light and the time course of its mRNA expression in response to insulin is prolonged (Kwak and Dixon, 1995; Groom et al., 1996). The upregulation of MKP-2 message in response to mitogenic stimuli is delayed and less pronounced compared to MKP-1. In addition, MKP-2 transcription is not induced by oxidative stress or heat shock (King et al., 1995; Guan and Butch, 1995; Misra-Press et al., 1995). VHR is unresponsive to extracellular stimuli, including oxidative stress or growth factor treatment (Kwak and Dixon, 1995). The cytosolic MKP-3 is not an immediate early gene; its message expression is weakly induced by serum and is unaffected by heat shock, oxidative stress, or UV light (Groom et al., 1996). In PC12 cells, NGF causes a robust upregulation of MKP-3, whereas in MM14 muscle cells, bFGF causes a similar induction (Muda et al., 1996b; Mourey et al., 1996). bFGF withdrawal from MM14 cells decreases MKP-3 expression, resulting in a subsequent increase in ERK activity, concomitant with differentiation (Mourey et al., 1996). MKP mRNA expression is regulated by mitogenic and stress-activated MAPK pathways. MKP-1 promoters contain Spl, NFl-like, AP2, AP1, Ebox, and CRE enhancer sites as well as a TATA basal promoter (Noguchi et al., 1993; Kwak et al., 1994). AP1 promoters are known to be regulated by ERK or JNWSAPK phosphorylation of c-Jun and CRE promoters can be regulated by JNUSAPK or p38 MAPK phosphorylation of ATF2 (see Section IV,A). Indeed, MKP-1 appears to be regulated by both ERKlI2 and JNWSAPK in a cell type-specific fashion. In NIH3T3 cells, activation of the JNWSAPK pathway induces MKP-1 gene expression whereas selective stimulation of the ERK pathway inhibits MKP-1 transcription (Bokemeyer et al., 1996). Thus, MKP-1 mediates cross-talk between ERK and JNWSAPK pathways in this system because JNWSAPK induces MKP-1, which in turn inactivates ERK (Bokemeyer et al., 1996). In contrast, in CCL39 fibroblasts, activation of ERK induces MKP-1 and MKP-2 expression, forming a negative feedback loop, whereas agonists that activate JNK have no effect on MKP-

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1 or MKP-2 (Brondello et al., 1997). The incongruous results in these studies necessitate further investigation into the role of MAPKs in MKP-1 regulation. Similar to MKP-1, the PACl promoter contains an AP2 site and an E-box element. PACl is positively regulated by the ERK pathway in mouse hematopoietic cells, constituting another inhibitory feedback loop (Grumont et al., 1996). Activation of yeast pheromone response and stress-activated MAPK pathways is also attenuated by the feedback induction of PTPs and DSPs (see Section VI). Thus, it is apparent that negative feedback loops are a conserved mechanism for MAPK pathway downregulation.

B. Serinenhreonine Phosphatases Dual specificity MKPs are not the exclusive regulators of MAPK pathways, as serinekhreonine protein phosphatases 1 (PPl) and 2A (PP2A) negatively regulate both MKKs and MAPKs. PP1 and PP2A inactivate ERKl and ERK2 and MKKl and MKK2 in vitro by dephosphorylation of regulatory Ser/Thr residues within their activation lip (Anderson et al., 1990; Ahn et al., 1991,1993; G6mez and Cohen, 1991; Nakielny et al., 1992). In addition, treatment of cells with okadaic acid, a potent inhibitor of PP1 and PP2A, activates the ERK pathway (Haystead et af., 1990; Gotoh et af., 1990b; Casillas et af., 1993). Overexpression of SV40 small T antigen, which interacts with and inhibits PP2A, stimulates MKKl and ERK activity while having no effect on Raf-1 activity in CV-1 cells (Sontag et al., 1993).In PC12, PAE, or 3T3-Ll cells, the rapid (15 min) inactivation of ERK following growth factor stimulation occurs independently of MKP-1 expression (Wu et al., 1994; Alessi et al., 1995b). In these cells, PP2A is the major phosphatase acting on ERK and MKKl, functioning coordinately with an unidentified Tyr,,,-specific phosphatase.(Alessi et al., 1995b).PP2A is negatively regulated by phosphorylation of its catalytic subunit (Tyr307)by receptor and nonreceptor tyrosine kinases (Chen, J. et al., 1992, 1994). Furthermore, phorbol ester stimulation of ventricular cardiomyocytes transiently suppresses PP2A activity, correlating with phosphorylation of the PP2A catalytic subunit (Quintaje et al., 1996). This temporary decrease in PP2A activity is accompanied by a transient increase in cytosolic ERK activity, indicating that PP2A is a physiological regulator of ERK.

C. Protein Tyrosine Phosphatases Tyrosine-specific phosphatases also attenuate signaling in MAPK modules. S . cerevisiae and S . pombe utilize PTPs in coordination with PP2C-related PTCs to regulate stress response pathways (Table I). In mammals, selective

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dephosphorylation of Tyrlss on ERK2 by the transmembrane PTP, CD45,is completely inactivating (Anderson et al., 1990). In general, a well-defined PTP that regulates ERK physiologically has yet to be reported in vertebrate systems, although one ERK-inactivating PTP has been purified to near homogeneity from Xenopus eggs (Sarcevic, 1993). Such an enzyme would probably act in conjunction with PP2A, efficiently dephosphorylating Tyrl8? following the rate-limiting removal of Thr,,, phosphoester by PP2A (Alessi et al., 1995b). The advantage of MAPK regulation by dual specificity phosphatases vs tyrosine-specific phosphatases probably resides in substrate specificity. For example, dual specificity VHR preferentially recognizes diphosphorylated substrate in an ordered mechanism that entails rapid removal of phosphotyrosine followed by slow hydrolysis of phosphothreonine (Denu et al., 1995b). Thus, DSPs do not require the initial action of a serinekhreonine phosphatase in order to efficiently dephosphorylate phosphotyrosine.

IV. CELLULAR SUBSTRATES OF MAP KINASES In uitro, the only targets for MKKs that have been identified are MAP kinases, therefore, specificity of downstream signaling in MKWMAPK cascades appears to be controlled at the level of ERK, JNWSAPK, and p38 MAPK. ERK favors substrates containing Pro residues at the P+ 1 position, with secondary preference for Pro at the P-2 position (Alvarez et al., 1991; Davis, 1993). JNWSAPK and p38 MAPK also recognize Pro at P+1, although the sequence determinants that confer differential recognition by ERK, JNK, or p38 MAPK have not been reported. In uitro targets for MAP kinases include nuclear transcription factors, metabolic enzymes, cytoskeletal proteins, and other signaling components (Table 11). Many of these also appear to be physiological targets, based on peptide map comparisons between in uitro vs in uiuo phosphorylation sites. Important reagents have become available that enable further correlation between kinase activation and substrate phosphorylation in response to various stimuli. Constitutively active mutants of Raf and MKKs have been expressed or microinjected into cells to activate signaling through various MAP kinase pathways. A particularily useful construct fuses the N-terminally truncated Raf-1 catalytic domain to the steroid-binding domain of the estrogen receptor (McCarthy et al., 1995). This chimera can be activated within minutes of adding estradiol to cells and has been used to measure effects of acute signaling through the Raf/MKK/ERK pathway (Pumiglia and Decker, 1997; Bianchini et al., 1997). Active MKKl and MKK2 mutants have been designed with activities ranging from 10-fold over basal to those comparable

OD

N

Table 11 Substrates for MAP Kinases and MAPKAP Kinases Substrate

Function

A. ERK 112 substrates 1. Protein kinases Rsk-1, Rsk-2 Rsk-3 MAPKAP kinase 2

MAPKAP kinase 3 Mnkl, Mnk2 2. Transcription factors ~62~~~Elk-2 SAP-la, SAP-2 ER8 1 Pointed P2 (Drosophila) Yan (Drosophila) ERF PPAR-71 Estrogen receptor C-FOS Fra-1, Fra-2 c-Jun JUN (Drosophila) C-MYC N-Myc HSF-1 GATA-2 TAL-1/SCL NF-IL6

Sequence

LLMT,,PCYT,,ANFV PQPPT,,PALP NSLTT,,,PCY AIS,,,PGMK VF’QT,,,PLHTSR

Reference

Sutherland et al. (1993a) Y. ZHao et al. (1995) Ben-Levy et al. (1995)

Ludwig et al. (1996) Waskiewicz et al. (1997) SRF activation SRF activation Ets activation Ets repression Ets repression Ets repression Suppression adipogenesis Receptor activation Fos stabilization

STLS,,,PIAPRS,,,PAK STLS,,,PVAPLS,,,PAR

Inhibition DNA binding AP-1 activation Myc activation Myc repression Heat shock response Hematopoietic transcription TAL-1 activation Interleukin-6 signaling

TPPLS,,,PIDME VINS,,PD, TVNT,,PD LPPTS,,PSRR PLS,,PS PPSPPQS,,,PR

PLT15,PG GGPLT,,,PRRV PAS,,,P PPPQLS,,,PF DSLSS,,PTLL

MVQLS,,,PPAL SSPPGT,,,PSP

Cavigelli et al. (1995) Price et al. (1995) Janknecht (1996) Brunner et al. (1994a) Rebay and Rubin (1995) Sgouras et al. (1995) Hu et al. (1996) Kato et al. (1995) R. Chen et al. (1996) Gruda et al. (1994) Alvarez et al. (1991) Peveraii et al. ( 1996) Alvarez et al. (1991) Manabe et al. (1996) B. Chu et al. (1996) Towatari et al. (1995) Wadman et al. (1994) Nakajima et al. (1993)

NTF-1IElf-1 (Drosophila) Tristetraprolin AMLl P53 3. Signaling components SOSl

EGF receptor Phospholipase A2 HSPDE4B2B Rab4 Inhibitor-2 PTP-2C 4. Cytoskeletal proteins MAP-2 Caldesmon Dystrophin Synapsin I Tau 5. Other targets Tyrosine hydroxylase Connexin-43 Stathmin Oncoprotein M Topoisomerase I1 Q

Torso response element Early response gene Acute myeloid leukemia Checkpoint control p2lRas activation

EGF signaling PLA, activation CAMPphosphodiesterase Glucose transport PP1 activation PTPase inhibition Microtubule association Smooth muscle contraction Cytoskeleton organization Neurotransmitter release Microtubule association Neurotransmitter synthesis Gap junction communication Microtubule polymerization DNA supercoiling

PLS,,,PS QIQPS,,,PPWS GSIAS,,,PSVH TET,,PGP, PAT,,PW GPRS ,,AS AESS,,,,PS HLDS,,,,PPA PRYS,,,,IS VEPLT,,,PSGEA SYPLS,,,PLSD 1pQs48,ps,,9ppL

QLRS,,,PRTT PST,PY

NKS,,,PAPK VTS,,PTK PVAS,,PAAPS6, PGS PPAS,,,PSPQ YSS199PGS202P

AIMS,,PRFK PLS,,,PSK PLS,,9PMS,,,PP ILS,,PRS, PLS,,PP LILS,,PRSK LPS,,,,PRG

Liaw et al. (1995) Taylor et al. (1995) Tanka et al. (1996) Milne et al. (1994) Corbalan-Garcia et al. (1996)

Northwood et al. (1991) Lin et al. (1993) Lenhard et al. (1996) Cormont et al. (1994) Q. Wang et al. (1995) Peraldi et al. (1994) Ray and Sturgill (1987) Adam and Hathaway (1993) Shemanko et al. (1995) Jovanovic et al. (1996) Drewes et al. (1992) Haycock et al. (1992) Warn-Cramer et al. (1996) Leighton et al. (1993) Marklund et al. (1993) Wells and Hickson (1995) (continues)

Table 11.

(continued)

Substrate

Function

Sequence

B. J W S A P K substrates MAPKAP kinase 3 ~62'~~Elk-l P53 Neurofilament H c-Ju~ C. p38 MAPK substrates MAPKAP kinase 2 MAPKAP kinase 3 ATF2 CHOP(GADD 153) Phospholipase A2 Max D. RSK1,2 substrates GSK-3 (Y GSK-3 /3 Glycogen targeting subunit L1 Tyrosine hydroxylase Nur77 C-FOS SRF CREB CBP E. MAPKAP b a s e 2 substrates hsp27 Tyrosine hydroxylase LSP-1 F. MAPKAP kinase 3 substrate Hsp27

SRF activation

Neurofilamentpolymerization AP-1 activation

STLS,,,PIAPRS,,,PAK VTET,,PGP APAT,,PW KSPXE repeats LLTS,,PDVGLLKLAS,,PEL LTT,,,PCY,

ATF2 activation CEBP activation

PQT,,,PL

DQT,9PT,1PT

RTSQS,,PHS,,PDSS

Myc activation

PP1 activation Neural cell adhesion DOPA synthesis Transcriptional regulation Fos stabilization SRF activation CREB activation Transcriptional coactivation

RARTSS,,FAEP RPRTTS,FAES RRGS,,ESS KGGKYS,,,,VK RRQS,,LIE AHRKGS,,,SSN RSLS,,,EM RRPS1,,YR

Reference Ludwig et al. (1996) Cavigelli et al. (1995) Milne et al. (1995) Giasson et al. (1996) Pulverer et al. (1991) Engel et al. (1995a) Ludwig et al. (1996) Gupta et al. (1995) Wang and Ron (1996) Kramer et al. (1996) Zervos et al. (1995) Sutherland and Cohen (1994) Stamhlic and Woodgett (1994) Dent et al. (1990) Wong et al. (1996) Sutherland et al. (1993b) Chen et al. (1992) R. Chen et al. (1996) Chen et al. (1992) Xing et al. (1996) Nakajima et al. (1996)

Actin polymerization DOPA synthesis Actin binding

RQLS,,SSGV RRAVS,,ELD, RRQS,,LIE KSNS,,VKK, AVA!5252TKT

Stokoe et al. (1992b) Sutherland et al. (1993b) Huang et al. (1997)

Actin polymerization

RQLS,,SSGV

Clifton et al. (1996)

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to phosphorylated enzyme (Brunet et al., 199410; Huang and Erikson, 1994; Mansour et al., 1994; Cowley et al., 1994; Mansour et al., 1996b). Constitutively active mutants of MKK3 and MKK6 have also been developed by the substitution of regulatory phosphorylation sites with acidic amino acids (Xia et al., 1995; Raingeaud et al., 1996). Finally, a constitutively active mutant of ERK mutation (“sevenmaker,” D319N) was identified by its gain-offunction phenotype in Drosophila eye development (Brunner et al., 1994b). Although the mutant ERK appears to be regulated normally by phosphorylation, its elevated activity is conferred by its greater resistance to dephosphorylation and inactivation by several protein phosphatases (Bott et al., 1994; Chu, Y. et al., 1996; Oellers and Hafen, 1996). This mutant has been used with variable success in mammalian systems due to its low level of activity (three- to fourfold). Several inhibitors have been useful for defining the involvement of various MKKs or MAPKs in signaling responses. Catalytically inactive mutants of MKKs 1-4 and 6, ERKs 1 and 2, JNK/SAPK, and p38 MAPK have been reported to work as dominant negative regulators of signaling, presumably through nonproductive interactions with upstream activators or downstream targets (Alawi et al., 1993; Frost et al., 1994; Zanke et al., 1996; Raingeaud et al., 1996). Inhibition of signaling has also been achieved using full-length antisense transcripts or antisense oligonucleotides directed toward Raf-1 or ERK (Pa& et al., 1993; Sale et al., 1995; Muthalif et al., 1996). PD98059 is a cell-permeable inhibitor selective for MKKl and MKK2, which blocks MKK activation at concentrations ranging from 10 to 100 p M (Dudley et al., 1995; Alessi et al., 1995a). In vitro, the IC,, for PD98059 ranges from 2 p M (for MKK1) to 50 p M (for MKK2). Antiinflammatory drugs, SB203580 and SB202190, are inhibitors of p38 MAPK a and p (but not y or S ) , with IC,, -1 pM. (Lee et al., 1994; Bayaert et al., 1996). Using these reagents, the involvement of different MKKs or MAPKs in various cellular responses can be established by correlating dose-response curves for signaling inhibition vs kinase inhibition.

A. Protein Kinase Substrates for MAPKs Five protein kinases can be activated by ERK phosphorylation and thus mediate MKWERK signaling, referred to as MAPK-activated protein (MAPKAP) kinases 1, 2, and 3 and Mnks 1 and 2. MAPKAP kinase-1 (Y and p were first described as ribosomal S6 kinases, pp90 Rsk-1 and Rsk-2 (Sturgill et al., 1988), which are activated through phosphorylation at two threonine residues within the activation lip (Sutherland et al., 1993a; Grove et al,, 1993). However, although ribosomal protein S6 was the substrate first used to identify Rsk activity, S6 appears to be a physiological target not of Rsk,

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but of p70 S6 kinase (Chung et af., 1992).Another homolog, Rsk-3, is phosphorylated but not activated by ERK2, suggesting that ERK may be needed but is not sufficient for activation (Zhao, Y. et af., 1995).MAPKAP kinase-1 isoforms each have two consensus kinase catalytic domains, both which are functional, with ERK phosphorylation elevating the activity of the C-terminal domain (Fisher and Blenis, 1996). These enzymes target several substrates for phosphorylation, with specificity for Arg at position P-3 (Stokoe et al., 1993). The glycogen-associated targeting subunit of PP1 is phosphorylated by ISPK, a protein kinase with properties similar to MAPKAP kinase-1, conferring activation of PP1 in response to insulin (Lavionne et af., 1991; Begum, 1995). Glycogen synthase kinase-3, a protein kinase involved in glycogen synthesis as well as cell fate determination, is inactivated in vitro and in intact cells on phosphorylation by MAPKAP kinase-la (Stambolic and Woodgett, 1994; Sutherland and Cohen, 1994; Eldar-Finkelman et al., 1995). Some substrates for MAPKAP kinase-1 are involved in transcription, including CAMP response element-binding protein (CREB)(Ginty et al., 1994; Xing et al., 1996),CREBbinding protein (CBP) (Nakajima et af., 1996), c-fos, Nur77, and serum response factor (Chen, R. et al., 1992,1996). Thus activation of RsUMAPKAP kinase-1 by ERK provides a means of gene regulation. Genetic lesions in MAPKAP kinase-1 p are implicated in Coffin-Lowry syndrome (Trivier et al., 1996), indicating a role for Rsk in human developmental diseases. MAPKAP kinase-2 is a 40-kDa enzyme, which in addition to a single catalytic domain contains a C-terminal autoinhibitory domain (Zu et al., 1995) and an N-terminal proline-rich region that interacts with SH3 domaincontaining proteins (Engel et af., 1993; Plath et af., 1994).Activation occurs through phosphorylation within the catalytic as well as the autoinhibitory domain (Ben-Levy et al., 1995; Engel et al., 1995a). MAPKAP kinase-2 is phosphorylated by both p38 MAPK and ERK, although p38 MAPK is probably the more important physiological regulator because many growth factors that strongly activate ERK do not activate MAPKAP kinase-2 (Stokoe et al., 1992a; Rouse et al., 1994). Once activated, MAPKAP kinase-2 recognizes substrate specificity determinants of Arg at P-3 and hydrophobic residues at P-5 (Stokoe et al., 1993). MAPKAP kinase-3 (also called chromosome 3p kinase, 3pK) is a 42-kDa enzyme identified in a genomic locus frequently disrupted in small cell lung cancers (Sithanandam et al., 1996). In vitro, MAPKAP kinase-3 can be activated by ERK, p38 MAPK, and JNWSAPK (Ludwig et al., 1996; McLaughlin et al., 1996) and is thus an example of a substrate targeted by all three pathways. Both MAPKAP k'inase2 and MAPKAP kinase-3 are activated by cellular stresses and inflammatory cytokines, and both enzymes phosphorylate heat shock protein 2 7 (hsp27) (Engel et al., 1995b, McLaughlin et al., 1996; Clifton et al., 1996). hsp27 behaves as an actin capping protein, thus MAPKAP kinases 2 and 3 are potential regulators of cytoskeletal assembly.

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M n k l and 2 were identified in screens for ERK interacting proteins (Waskiewicz et al., 1997; Fukunaga and Hunter, 1997). Both enzymes can be directly activated by ERK or p3 8 MAPK phosphorylation. Once activated, M n k l and 2 phosphorylate eukaryotic initiation factor 4E (Waskiewicz et al., 1997) suggesting a mechanism for translational control by these two MAPK pathways.

B. Nuclear Transcription Factors MAP kinases phosphorylate several proteins belonging to the Ets family of helix-turn-helix transcription factors. The p62 ternary complex factor ( ~ 6 2 ~ ~ ~ Aregulates 4 k - l ) serum response element (SRE)transcription through its interaction with serum response factor (SRF) (Shaw et al., 1989; Graham and Gilman, 1991). Transactivation is potentiated by phosphorylation of ~ 6 2 ~ ~ ~ / Eby l kERK, - 1 JNK/SAPK, or p38 MAPK (Marias et al., 1993; Janknecht et al., 1993; Gille et al., 1992, 1995a,b; Cavigelli et al., 1995; Whitmarsh et al., 1995; Price et a/., 1996). Ternary complex factors Sapl and Net/Erp/Sap2, which also interact with SRF and SRE, are substrates for ERK and p38 MAPK; Sapl is also a substrate for JNK/SAPK (Price et al., 1995, 1996; Janknecht et al., 1995). Transactivators ERM/ER81 and most likely Etsl and Ets2 are phosphorylated by ERK (Janknecht, 1996; Janknecht et al., 1996; Conrad et al., 1994). Ets family members that function as transrepressors are also directly regulated through phosphorylation by ERK. An Ets2 repressor factor (ERF), similar to p62TCF/Elk-l in its DNA-binding domain, is phosphorylated and inhibited in response to vRaf in mammalian cells (Sgouras et al., 1995). In vitro, ERF is phosphorylated by both ERK2 and cdc2 and is most likely regulated by phosphorylation at ThrS26, as substitution of this residue with Glu leads to loss of repression. Genetic screens for downstream transcriptional targets of ERK in C. elegans and Drosophila have so far identified mainly Ets family members as physiological substrates controlling developmental pathways (see Section V,B). MAP kinases also target bZIP transcription factors for phosphorylation and activation. Both JNWSAPK and p38 MAPK phosphorylate activating transcription factors ATFa and ATF2 (Gupta et al., 1995,1996; Bocco et al., 1996), members of the ATF/CREB family that heterodimerize with c-Jun, NF-KB, and CREB. Phosphorylation enhances ATF2 transcriptional activity, whereas mutation of phosphorylation sites or expression of dominant negative mutant JNK/SAPK inhibits transcription (Gupta et al., 1995).Dominant negative ERK mutants block transactivation by AP-1, showing that ERK activation is necessary for AP-1 transcriptional function (Frost et al., 1994). Although ERK was originally found to phosphorylate c-Jun at Ser,,

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and Ser,, within its activation domain (Pulverer et al., 1991), later studies showed that ERK also phosphorylates residues at the C terminus of c-Jun, which may lead to negative regulation through inhibition of DNA binding (Alvarez et al., 1991; Chou et al., 1992; Minden et al., 199413).JNWSAPK is the more likely physiological c-Jun kinase in mammalian systems because it forms a tight complex with c-Jun and phosphorylates it more efficiently (Hibi et al., 1993; Minden et al., 1994b; Dirijard et al., 1994).In agreement, in vivo studies show a stronger correlation between c-Jun phosphorylation and JNWSAPK activation than ERK activation (Smeal et al., 1994; Westwick et al., 1994). However, it is still possible that ERK activates AP-1 through direct phosphorylation of c-Jun under some conditions. For example, regulation of photoreceptor differentiation in Drosophila involves c-Jun phosphorylation at Ser,, and Ser9,, dependent on the activity of Rolled (Treier et al., 1995; Peverali et al., 1996). Drosophila JNWSAPK is not required for photoreceptor differentiation, indicating that ERK is the physiological regulator of c-Jun in this system. In vitro, Fos, Fral, and Fra2 are potential targets for direct phosphorylation by ERK, as well as MAPKAP kinase-1, and sites phosphorylated on these transcription factors in vitro are similar to those observed in vivo (Chen et al., 1993; Gruda et al., 1994). In intact cells, phosphorylation of Fos in response to ERK stimulation leads to enhanced Fos protein stability (Okazaki and Sagata, 1995a). A serum-stimulated Fos kinase (FRK) distinct from ERK was shown to activate AP-1 transactivation and may represent an equally important regulator of Fos (Deng and Karin, 1994). c-Myc is phosphorylated by ERK at Ser6,, the primary site for phosphorylation in vivo (Alvarez et al., 1991; Davis, 1993). This residue may be important for transcriptional regulation (Seth et al., 1992), although conclusive evidence for transactivation through ERK is not available. In cell extracts, ERK binds tightly to the N terminus of c-Myc (Gupta and Davis, 1994), an interaction that is disrupted by Myc phosphorylation, suggesting functional interactions between enzyme and substrate. Another member of the Myc family, the N-Myc repressor, is phosphorylated by ERK at Ser,,. This phosphorylation event may be required for N-Myc function because mutation of Ser,, results in loss of repression (Manabe et al., 1996). ERK is also involved in steroid hormone receptor signaling, as demonstrated by its regulation of receptors for estrogen and peroxisome proliferator-activated receptor-y (PPAR-y). Estradiol activates ERK, most likely mediated through tyrosine phosphorylation of Shc (Migliacio et al., 1996). Transactivation of estrogen-response element/luciferase reporters by EGF is dependent on Ras and MKK and can be inhibited by MAP kinase phosphatase-1 expression, suggesting that transcriptional activity is mediated through ERK (Kato et al., 1995; Bunone et al., 1996). Correspondingly, estrogen receptor is phosphorylated by ERK at Ser,,, in vitro; this residue is

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necessary for optimal transcription because its mutation leads to decreased response. In uiuo, however, Serlla can be phosphorylated in the absence of estradiol, suggesting that ERK either mediates steroid-independent transactivation or coregulates steroid-dependent activation (Arnold et al., 1995; Bunone et al., 1996). The analogous residue (Serl12)on the related PPAR-.I, a transcriptional regulator controlling adipocyte differentiation, is phosphorylated in response to EGF, phorbol esters, serum, or insulin (Hu et al., 1996). Phosphorylation at Ser, 12 by ERK blocks transcriptional activity of PPAR-y, providing a means by which growth factors may block adipogenesis through MKKERK signaling.

C. Signaling Components Cytosolic substrates for MAP kinases include signaling components that may be involved in feedback regulation or cross-regulation of other pathways. A likely target for feedback inhibition by ERK is the Ras guanine nucleotide exchange factor, Sosl. In uitro, Sosl is highly phosphorylated by ERK, leading to a characteristic retardation in gel mobility that is also observed in factor-stimulated cells (Cherniack et al., 1994; Buday et al., 1995). Peptide mapping studies have identified at least five residues on hSosl that are phosphorylated by ERK, four of which are phosphorylated in uivo following growth factor stimulation (Corbalan-Garcia et al., 1996). Interestingly, not all of these sites conform to the consensus Sermhr-Pro recognition motif for the MAP kinases (Table 11), suggesting some deviation in ERK substrate specificity. Disruption of the Sosl-Grb2 complex is sensitive to PD98059, indicating a functional role for MKKl in Sos regulation (Dong et al., 1996; Holt et al., 1996). Part of this effect is insensitive to MKP-1 and may thus represent a unique example of ERK-independent MKKl signaling. Phosphorylation of Sosl following cell stimulation correlates with the destabilization of interactions among Sosl-Shc, Sosl-EGFR, and Sosl-Grb2 (Rozakis-Adcock et al., 1 9 9 9 , thus interfering with Ras/Raf activation. Thus, through this mechanism, ERK may serve as its own negative regulator. Negative feedback regulation by ERK has also been proposed to occur through ERK phosphorylation of EGF receptor at Thr,6, (Morrison et al., 1996). However, the effect of phosphorylation at this site is ambiguous, as its mutation to Cys had no effect on receptor autophosphorylation or downregulation of EGF signaling. MAP kinases also phosphorylate enzymes involved in eicosanoid biosynthetic pathways. Cytosolic phospholipase A, (cPLA,) is the rate- limiting enzyme in pathways of agonist-stimulated arachidonic acid release. In vitro, ERK phosphorylates cPLA, on Ser,,,.-, resulting in elevated activity (Lin et al., 1993; Nernenoff et al., 1993; Gordon et al., 1996). Temporal correla-

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tions between the agonist stimulation of ERK and Ser,,, phosphorylation versus the activation of cPLA, and its translocation to the plasma membrane support the importance of ERK as a physiological regulator of cPLA, (Durstin et al., 1994; Sa et al., 1995). Furthermore, in Madin-Darby canine kidney cells, activation of arachidonic acid release in response to stimulation of or,-adrenergic receptors by epinephrine is inhibited by PD98059 (Xing and Insel, 1996). However, cPLA, can be regulated by other kinase cascades as well, depending on the cell type. For example, thrombin stimulation of cPLA, in platelets is insensitive to PD98059, but is completely blocked by SB203580, implicating a pathway mediated by p38 MAPK (Kramer et al., 1996). Another potential target in eicosanoid signaling is cyclooxygenase2 (COX-2) which is transcriptionally upregulated in response to all three MAPK pathways (A. Sorokin, personal communication). In addition, membrane translocation and activation of 5-lipoxygenase in response to the Ca2+ionophore have been shown to be inhibited by PD98059 (Lepley and Fitzpatrick, 1996), implicating ERK in the regulation of leukotriene synthesis.

D. Cytoskeletal Proteins Earlier studies demonstrated that addition of active ERK to cell-free extracts of interphase Xenopus oocytes resulted in microtubule polymerization resembling an M-phase array (Gotoh et al., 1991), suggesting a role of MAPK in meiotic spindle formation. Association of ERK with microtubule networks, determined by indirect immunofluorescence in mammalian cells has also been reported (Reszka et al., 1995), although this is may be due to high levels of ERK expression in cells. Several cytoskeletal substrates for MAP kinases are implicated in neuronal function. ERK was first described as a protein kinase that phosphorylates microtubule-associated proteins 1 and 2 (Hoshi et al., 1988; Ray and Sturgill, 1987), although the physiological significance of these phosphorylation events has not been resolved. The microtubule-binding protein, Tau, is highly phosphorylated by ERK, as well as by other proline-directed (GSK3, cdk5) and nonproline-directed (CAMP-dependent kinase, protein kinase C, casein kinase I, CaM kinase 11)protein kinases (Hosoi et al., 1995; Singh et al., 1996). ERK phosphorylation of Tau resembles an abnormal phosphorylation pattern associated with Alzheimer’s disease and inhibits microtubule-Tau interactions (Drewes et al., 1992). However, cotransfection of Tau with Raf-1 in COS cells does not result in Tau phosphorylation (Latimer et al., 1995), suggesting that ERKs may not be as important as other proline-directed kinases. The neuronal intermediate filament, neurofilament-H, is phosphorylated by JNWSAPK at repeated Lys-Ser(P)-Pro-X-Glu motifs

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within the C-terminal tail domain (Giasson et al., 1996). Hyperphosphorylation of neurofilament H occurs in response to stress activation of JNWSAPK in PC12 cells and dorsal root ganglia and is a characteristic feature of several neurodegenerative diseases. Synapsin I, an actin-binding protein involved in cross-linking synaptic vesicles with cytoskeleton, is phosphorylated concomitantly with ERK activation in response to neurotrophins (Matsubara et al., 1996; Jovanovic et al., 1996). Phosphorylation by ERK at Ser,-, Ser,-, and Ser,,, of synapsin I reduces its F-actin bundling activity in vitro, whereas other proline-directed kinases had no effect.

V. RESPONSES TO MAPK PATHWAYS: GROWTH AND DIFFERENTIATION A. Regulation of Cell Growth and Transformation The rapid activation of Raf-1, MKK1/2, and ERK1/2 observed in response to mitogen stimulation implies that this pathway is necessary for cell cycle progression through G1. This is supported by evidence showing elevated DNA synthesis or cell growth under conditions of ERK activation such as expression of constitutively active mutant MKKl (Brunet et al., 1994; Seger et al., 1994). Correspondingly, mitogen-induced DNA synthesis is blocked by antisense RNA-mediated ERK downregulation (Pa& et al., 1993), dominant negative ERK mutants (Frost et al., 1994; Troppmair et al., 1994), or treatment of cells with the MKK inhibitor, PD98059 (Dudley et al., 1995; Seufferlein et al., 1996). In many cells, ERK activation occurs within minutes following factor treatment and declines after 1-2 hr. However, the kinetics can be variable under different conditions, raising the possibility that different responses may depend on the time frame of activation. For example, responses of CCL39 fibroblasts to thrombin, which is mitogenic, vs thrombin proteolytic peptide, which is nonmitogenic, respectively, occur with sustained vs transient kinetics of ERK activation (Vouret-Craviari et al., 1993), suggesting that persistent activation of ERK is required for cells to pass G1. Likely targets for regulation by ERK during G1 are immediate early genes such as Fos and Egr-1, which can be induced through ~ 6 2 ~ ~ ~ Ephoslk-l phorylation and activation of serum response factor promoter elements (Kortenjann et al., 1994; Hipskind et al., 1994a,b; Beno et al., 1995). Cyclin D1 is transcriptionally induced by MKKERK, and provides a link between the ERK pathway and S-phase entry through regulation of cyclidcdk activity (Lavioe et al., 1996).Phosphorylation of eIF4E by Mnk may also promote translation of key proteins needed for S phase entry (Waskiewicz et al., 1997). Although the best documentation of Raf/MKKERK activation occurs in

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early G1, a few reports exist demonstrating Raf and ERK activation during mitosis in synchronized mammalian cells (Laird et al., 1995; Edelmann et al., 1996). Antibodies that specifically stain activated ERK show colocalization of ERK with spindle poles and condensed chromatin in mitotic cells (P. Shapiro and N. Ahn, unpublished results). In oocytes, ERK associates with microtubule organizing centers and is involved in meiotic spindle formation (Verlhac et al., 1993).Furthermore, activation of ERK by v-Mos or constitutively active MKKl leads to partial chromosome condensation and formation of novel germinal vesicle microtubule arrays (Choi et al., 1996b). These data suggest a role for ERK in microtubule rearrangements during M phase. Transfection of 3T3 cells with constitutively active mutant MKKl and MKK2 (Mansour et al., 1994a, 1996b; Brunet et al., 1994b; Cowley et al., 1994) demonstrated that MKKERK induces hallmarks of cellular transformation, such as high saturation density, anchorage independent growth, cell rounding, and solid tumor formation in mice. In addition, cell transformation induced by v-ras or v-mos can be blocked by dominant interfering mutants of MKKl or ERK 1 or 2 (Troppmair et al., 1994; Okazaki and Sagata, 1995b). Thus, sustained ERK activation most likely accounts for many of the cellular responses observed with oncogenic Ras, Raf-1, and Src. In somatic cells, elevated expression of v-Mos leads to irreversible growth arrest, apoptosis, and increased ploidy, effects that are blocked by p53 (Fukasawa et al., 1995, 1997). This has led to the suggestion that constitutive MKK/ ERK activation in the absence of p53 may enhance chromosome instability during cell transformation. Although transformation is clearly dependent on RaWMKKIERK activation, the degree of tumorigenicity observed with activated forms of Raf or MKK1/2 is low compared to that observed with oncogenic ras. Interestingly, foci formation in NIH3T3 cells induced by v-rafcan be synergistically enhanced to levels observed with v-ras by coexpression of GTPase-deficient mutants of Racl or RhoA (Qiu et al., 1995; Khosravi-Far et al., 1995).Furthermore, dominant negative mutants of Rac or Rho blocked transformation in response to v-ras. Similar effects are observed with PI3’K, acting upstream of Rac (Rodriguez-Viciana et al., 1997).Thus, activation of MKK/ ERK is necessary but not sufficient to account for effects of Ras transformation, which instead requires the coordinated activation of other Ras effectors to control cytoskeletal reorganization.

B. Regulation of Cell Differentiation and Development In contrast to the role of MKK1/2 and ERK1/2 in regulating cell growth, MAPK pathways also play an important role in regulating embryonic development and cell differentiation. This is best demonstrated in whole animal

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studies, where activation of MKK and ERK is essential for vulval development in C. elegans, photoreceptor cell specification and anterior-posterior body patterning in Drosophila, mesoderm induction during Xenopus laevis embryo development, and positive T-cell selection. In addition, MAPK cascades have been shown to regulate the differentiation of mammalian cell lines that undergo processes of commitment along neuronal, blood cell, or fat cell lineages. Such examples illustrate the ability of different cells to utilize identical pathways to control seemingly opposite responses of cell proliferation vs cell growth arrest and expression of lineage-specific genes. 1. WORM DEVELOPMENT

An ERK module is central to the process of vulval development in the C. elegans hermaphrodite. During vulval formation, a set of vulval precursor cells (VPCs) receive an inductive signal from the gonadal anchor cell (AC), directing those VPCs nearest the AC to adopt the vulval fate (1"or 2"). The pathways determining vulval fate have been analyzed genetically utilizing Vulvaless (Vul) and Multivulva (Muv) mutants (reviewed by Sundaram and Han, 1996). Characterization of these mutants has revealed a Ras-regulated MKWERK pathway analogous to that found in mammalian cells. The inductive signal is encoded by the lin-3 gene, which is homologous to mammalian EGF (Hill and Sternberg, 1992). This signal is likely received at the VPC through the let-23 receptor tyrosine kinase (RTK), a member of the EGF receptor subfamily (Aroian et al., 1990, 1994). High dosages of lin-3 result in 1"VPCs, whereas lower doses induce 2" VPCs, indicating that VPC fate is modulated by the strength of signaling through the pathway (Katz et al., 1995). LET-23 appears to be localized to VPC cell junctions by LIN-2 and LIN-7, membrane-associated guanylate kinase (MAGUK)-like proteins (Hoskins et al., 1996; Simske et al., 1996). This localization may serve to position LET-23 toward the AC, the presumptive source of LET-3. SEM-5 is an adaptor protein homologous to mammalian Grb2 (Clark et al., 1992), presumably binds activated LET-23 (Stern et al., 1993), and associates with an as yet unidentified guanine-nucleotide exchange factor. Further downstream is the let-60 gene, which encodes the C. elegans Ras protein (Han and Sternberg, 1990; Beitel et al., 1990). LET-60 mediates the activation of the LIN45 Raf homolog (Han et al., 1993), which in turn activates a downstream MKK referred to as MEK-2 (Wu et al., 1995; Kornfeld et al., 1995a). MEK2 functions upstream of the ERK homolog SUR-UMPK-1 (Wu and Han, 1994; Lackner et al., 1994). Downstream of SUR-UMPK-1 are putative transcription factors, LIN-1 and LIN-31 (Miller et al., 1993; Beitel et al., 1995),whose corresponding promoter elements have yet to be identified. lin1 is an Ets family member, containing multiple ERK consensus phosphorylation sites, which negatively regulates vulval development, and lin-3 1 is re-

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lated to the HNF-3/fork head family of DNA-binding transcription factors. A reasonable model analogous to the behavior of mammalian ERK is that these transcription factors may be direct substrates for SUR-l/MPK-1 phosphorylation, activated through let-60 signaling. A novel Raf-related kinase, KSR, has been identified genetically as an enhancer of let-60 ras signaling in the vulva1 development system (Sundaram and Han, 1995; Kornfeld et al., 1995b).KSR appears to function either downstream or in parallel to let-60. In support of the genetics, biochemical analysis of human KSR indicates that this component enhances signaling through accelerating MKK and ERK activation (Therrien et al., 1996). 2. FRUIT FLY DEVELOPMENT

a . T h e ERK Pathway in Eye Development A variety of developmental processes in Drosophila are regulated by receptor tyrosine kinases. The torso receptor tyrosine kinase organizes the formation of the anterior and posterior regions of the embryo, whereas the Drosophila EGF receptor (DER)executes multiple developmental functions, including establishment of dorsal-ventral polarity of the egg (Sprenger et al., 1989; Price et al., 1989). Similarly, cell fate determination of the R7 photoreceptor cell in eye development is regulated by the sevenless RTK (Sev) (Hafen et al., 1987).These receptor tyrosine kinases signal through common ERK pathway component genes, although they are differentially expressed and activated by distinct ligands (reviewed by Perrimon, 1994). The sevenless eye development pathway is well characterized and will be the focus of this discussion. The Drosophila compound eye is composed of approximately 800 ommatidia (unit eyes), each of which contain eight photoreceptor cells (Rl-R8). Photoreceptor differentiation occurs in an ordered sequence, first generating R8, followed by R2IR.5 and R3/R4, R1 and R6, and finally R7 (reviewed by Yamamoto, 1994). Differentiation of the R7 photoreceptor, studied extensively using both genetic and molecular approaches, has established the ERK pathway as the mediator of the developmental signal. Signaling is initiated at the R7/R8 cell interface, where the sevenless receptor tyrosine kinase of R7 interacts with its ligand, bride of sevenless (Boss), a transmembrane protein expressed on the surface of the R8 photoreceptor (Kramer et al., 1991). Upon dimerization and tyrosine autophosphorylation of Sev, the SH2-SH3 adaptor protein Drk(Grb2) binds Sev and recruits Drosophila Sos (Olivier et al., 1993; Simon et al., 1993), resulting in the activation of Rasl (Simon et al., 1991; Bonfini et al., 1992). Rasl activation is attenuated by the Ras GTPase, Gap1 (Gaul et al., 1992). Another potential adaptor protein, daughter of sevenless (Dos), functions upstream of Rasl. Dos contains an N-terminal pleckstrin homology domain and is a substrate of the PTPase,

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corkscrew (Csw) (Raabe etal., 1996; Herbst etal., 1996).Although the function of dos is unclear, genetic relationships and biochemical data are consistent with a model in which dephosphorylation of Dos by Csw activates the signaling pathway downstream of Sev, leading to Rasl activation. Following Rasl, Drosophila Raf-1 activates the Drosophila MKK1, D-MeWDsorl kinase (Dickson et al., 1992; Tsuda et al., 1993; Hsu and Perrimon, 1994; Lu et al., 1994).The Drosophila ERK homolog, Rolled, is then activated by D-Mek (Brunner et al., 1994b; Biggs et al., 1994). As discovered in C. eleguns, KSR was identified in genetic screens as an enhancer of signaling, functioning downstream or in parallel to Rasl (Therrien et al., 1995). Interestingly, the protein phosphatase PP2A has appeared in genetic screens, both positively and negatively regulating the pathway downstream of Rasl; however, substrates for Drosophila PP2A are unknown (Wassarman et al., 1996b).Thus, the fundamental ERK signaling pathway is conserved between invertebrates and mammals. Several transcription factors regulated by the sevenless signaling cascade have been identified as Ets family members. yan and pointed (pnt)have negative and positive regulatory roles, respectively, in R7 photoreceptor differentiation (Lai and Rubin, 1992; O’Neill et al., 1994; Brunner et al., 1994a). The repressor activity of yan is abrogated on activation of the sevenless signaling pathway. The yan protein has multiple ERK consensus phosphorylation sites, and ERK phosphorylation of Yan appears to decrease its stability and alter its subcellular localization (Rebay and Rubin, 1995). The pointed gene encodes two related proteins, pntPl and pntP2, which are products of alternative splicing. pntPl is a constitutively active transcription factor, whereas pntP2 is transcriptionally active only when phosphorylated at its single ERK phosphorylation site (O’Neill et al., 1994; Brunner et al., 1994a). Thus, yan and pointed have opposing transcriptional activities and are differentially regulated by activation of the sevenless signaling pathway. Drosophila Jun (DJun) is also required for Ras-induced R7 photoreceptor differentiation (Bohmann et al., 1994). DJun and Pointed synergistically activate transcription from an AP-1Ets promoter element, indicating functional cooperation between these two transcription factors (Treier et al., 1995). Further, yan antagonizes the enhanced Pointed/DJun transcriptional effect. DJun is a MAPK substrate, and its phosphorylation correlates with photoreceptor differentiation (Peverali et al., 1996). Downstream target genes upregulated by signaling through the sevenless pathway include phyllopod and seven in absentia (sina), which encode nuclear proteins (Carthew and Rubin, 1990; Dickson et al., 1995; Chang et al., 1995). The prosper0 gene, a putative transcription factor involved in nervous system differentiation, is also upregulated in response to sevenless activation, and this induction appears to be mediated by yan and pointed (Doe et al., 1991; Kauffmann et al., 1996). Intriguingly, Phyllopod is also able to upregulate

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prosper0 in coordination with Sina. Phyllopod forms a complex with Sina both in vitro and in vivo, and it is likely that the PhyllopodSina complex has transcriptional activity.

b. T h e JNWSAPK Pathway in Epithelial Cell Migration A second Drosophila MAPK pathway is important for dorsal closure and the insect immune response, representing a unique example of the role of the JNWSAPK pathway in development. Dorsal closure occurs at midembryogenesis and entails the dorsalward spreading of epidermal cells over the amnioserosa membrane until fusion occurs at the dorsal midline (Young et al., 1993). Interestingly, this developmental process is regulated by the JNW SAPK pathway (see Section 11,B). The loss of function hemipterous gene is defective in dorsal closure and encodes a Drosophila JNK kinase (Hep) that is homologous to mammalian MKK3 and MKK4 (Glise et al., 1995). Hep is able to phosphorylate and activate a JNWSAPK homolog, DJNK, encoded by the basket gene (Sluss et al., 1996). Embryos lacking DJNK or that express dominant negative Drosophila cdc42, an upstream GTPase, are also deficient in dorsal closure (Sluss et al., 1996; Riesgo-Escovar et al., 1996). Thus, JNWSAPK pathway components appear to be conserved between mammals and flies. A Drosophila MEKK, Pk92B, has been cloned (Wassarman et al., 1996a).Although its role in development has not been reported, it is reasonable to expect a function upstream of Hep. In addition, DJNK is activated on exposure of cultured Drosophila cells to endotoxic lipopolysaccharide. DJNK activation coincides with a marked induction of antibacterial immune response genes, indicating that the JNWSAPK pathway also mediates an immune response (Sluss et al., 1996). Both developmental and immune responses are likely to be dependent on activation of the DJun transcription factor. Like the ERK homolog, rolled, DJNK efficiently phosphorylates DJun, yet the two pathways appear to be distinct as DJNK is not required in eye development (Sluss et al., 1996; Riesgo-Escovar et al., 1996). Interestingly, the Hep-DJNK pathway upregulates the expression of the puckered gene, which encodes a MAPK phosphatase (Riesgo-Escovar et al., 1996). Hence, regulation through a negative feedback loop is another conserved feature of the pathway.

3. MESODERM INDUCTION IN Xeuopus EMBRYOS

A role for MKWERK in vertebrate development has been documented in the early process of mesoderm induction. MKK and ERK act as cytostatic factors following the fertilization of Xenopus oocytes when their activities drop (Haccard et al., 1993; Kosako et al., 1994); however, their activities appear again following the blastula stage (MacNicol et al., 1995). In Xenopus embryos, signals derived from the vegetal hemisphere, including activin and

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basic fibroblast growth factor (bFGF), diffuse into the animal pole, leading to changes in gene expression and morphogenesis. Treatment of explanted animal poles with various factors show that signaling through bFGF is a key regulator of mesodermal induction, synergistic with activin signaling (reviewed by Gotoh and Nishida, 1996). The importance of Ras and Raf was indicated by inhibition of mesoderm induction on microinjection of dominant negative mutants of these enzymes into animal pole explants treated with bFGF (Whitman and Melton, 1992; MacNicol et al., 1993; Gotoh et al., 1995b). bFGF, but not activin, leads to the stimulation of ERK (Graves et al., 1994), and the involvement of MKKERK in bFGF signaling was confirmed by the induction of mesoderm on injection of constitutively active MKK, ERK (sevenmaker), or STEll mutants into explants (Labonne and Whitman, 1994; Gotoh et al., 1995; Umbhauer et al., 1995). The resulting mesodermal tissue was capable of undergoing further morphogenesis and cardiac actin expression by the dorsalizing factor, noggin. Likewise, the bFGF-induced mesodermal development was inhibited by microinjection of MAP kinase phosphatase-1 . Thus, signaling through MKKERK appears to be sufficient to mimic the effects of bFGF and most likely targets early regulators of mesoderm development, such as the Xenopus Brachyury (Xbra) immediate early gene. 4. NEURONAL DIFFERENTIATION

One approach to understanding the processes of differentiation at a mechanistic level is by manipulating cell lines that differentiate in culture. The rat pheochromocytoma (PC12) line is a widely used model for neuronal differentiation, in which nerve growth factor (NGF) or bFGF cause cell cycle arrest in G1, cytoskeletal rearrangements leading to neurite outgrowth, and expression of neuronal markers. Inhibition of MKKlI2 with PD98059 suppresses NGF-induced neuronal differentiation (Pang et al., 1995), and expression or microinjection of constitutively active MKKl induces neurite outgrowth in the absence of NGF (Cowley et al., 1994; Fukuda et al., 1995). The inhibition of cell proliferation in response to NGF is mediated through the induction of p21(Cipl/Wafl) and suppression of cyclidcdk activity (Pumiglia and Decker, 1997). This is blocked by PD98059, indicating that MKK/ERK induces G1 cell cycle arrest through control of p21(Cipl/Wafl) transcription. Under certain conditions, positive correlations between ERK activation and differentiation are not observed. For example, activation of ERK by mutant NGF or PDGF receptors does not necessarily lead to cell differentiation (Peng et al., 1995; Vaillancourt et al., 1995), and induction of cell differentiation by bone morphogenetic protein or activin A does not require ERK activation (Iwasaki et d.,1996). Therefore, the sufficiency of MKK/ERK activation in regulating PC12 cell differentiation is debatable.

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Nevertheless, MKWERK appears to play an essential role in controlling neuronal differentiation in response to NGF or bFGF signaling. An interesting feature of PC12 cells is that ERK is activated in response to both proliferative and differentiative stimuli. Several laboratories have reported short-term activation of ERK, which is downregulated within a few hours following treatment of cells with proliferation factors vs prolonged activation of ERK on treatment with differentiation factors (Gotoh et al., 1990a; Qiu and Green, 1992; Traverse et al., 1992).This prolonged activation can last up to 10 days and appears to be necessary to maintain the differentiated state and cell survival. The observed variation in kinetics of ERK activation has suggested that sustained activation of ERK may be necessary to trigger a late event necessary for differentiation, but not proliferation, of PC12 cells. A similar model has been proposed to explain proliferative responses in CCL39 cells (Vouret-Craviari et al., 1993; see Cell Growth above). In both cases, nuclear translocation of ERK occurs only in response to prolonged activation (Traverse et al., 1992; Vouret-Craviari et al., 1993), suggesting that the late event responsible for the differentiation of PC12 cells or mitogenesis of CCL39 cells involves the phosphorylation of nuclear targets.

5. BLOOD CELL DIFFERENTIATION MAPK activation occurs as an acute response in lymphoid cells to T-cell receptor or B-cell receptor stimulation (Whitehurst et al., 1992; Izquierdo et al., 1993; Tordai et al., 1994; Nel et al., 1995). In mature T cells that respond to TCR stimulation by transcription of interleukin-2, proliferation, or apoptosis, MAPKs are stimulated through a pathway involving coupling of the TCR-CD3 complex to Ras and Raf, mediated by Lck and ZAP70 tyrosine kinase interactions with TCR. ERK and JNK/SAPK activation appear to be involved in the transcriptional induction of interleukin-2, and TCR signaling is blocked by dominant negative mutant MEKKl in these pathways (Faris et al., 1996). Inhibition of IL-2 transcription also correlates with suppressed ERK and JNK stimulation following T-cell anergy produced in the absence of receptor costimulation (Li et al., 1996). Immature CD4-/CD8- T cells respond to TCR stimulation by positive selection pathways, controlling T-cell differentiation into CD4+/CD8+cells, or negative selection, controlling cell death. The involvement of MKWERK in TCR-regulated pathways was investigated by targeting a dominant negative mutant MKKl for expression in T cells under control of the Ick proximal promoter (Alberola-lla et al., 1995). Mice expressing this mutant showed lowered levels of CD4'8- or CD4-8' cells, indicating a requirement for MKKl during maturation of double-positive to single-positive thymocytes. No effects on negative T-cell selection or TCR-induced proliferation or apop-

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tosis were observed, suggesting an important role of alternative signaling pathways, such as those involving stress-activated kinases (Sen et al., 1996). Results consistent with these studies were observed in fetal thymic tissue utilizing retroviral transfection of MKKl into organ cultures (Crompton et al., 1996). Both double-positive and single-positive T cells were increased following transfection of constitutively active MKKl into tissue from TCRadeficient mice, and transfection of dominant negative MKKl blocked differentiation. No evidence for an effect of MKKl on T-cell proliferation was observed, in agreement with the whole animal study. A role for MKKERK in the differentiation of other blood cell types has also been examined by expression of constitutively active MKK mutants. A late event in blood cell differentiation is the commitment of nonlymphoid cells into megakaryocyte versus erythroid lineages. The human erythroleukemia cell lines, K562 and CMK, retain the capacity to differentiate into megakaryocytes in response to phorbol ester. In either cell type, expression of constitutive active MKKl leads to inhibition of cell growth, induction of characteristic megakaryocytic morphology, including increased cell size and multinucleation, and cell surface expression of integrin aIIbP3, an adhesion receptor specifically expressed in platelets (Whalen et al., 1997; Vik et al., 1997). In addition, the expression of globin genes in K562 cells is blocked by elevated MKWERK activity and is upregulated by the PD98059 inhibitor (Whalen et al., 1997), thus MKWERK promotes megakaryocyte differentiation at the same time it suppresses erythroid differentiation. These results illustrate an example of how MKKERK regulates lineage commitment in pluripotent cells at the level of marker gene expression.

6. ADIPOCYTE DIFFERENTIATION Adipocyte differentiation from fibroblasts involves a series of transcriptional events leading to growth arrest and induction of specific genes required for fat synthesis and degradation (reviewed by MacDougald and Lane, 1995). The 3T3-Ll cell line is a useful model that proliferates with a fibroblast morphology, but can be induced to differentiate into adipocytes by continuous exposure to insulin-like growth factor 1, glucocorticoid, fatty acids, and agents that elevate CAMP. Earlier studies showed induction of differentiation by expression of v-ras or v-rafin 3T3-Ll fibroblasts (Benito et al., 1991; Porras et al., 1994). The evidence implicating ERK in this process is conflicting, however. In one study, ERK antisense oligonucleotides, used to inhibit ERK activation in response to insulin, successfully blocked insulin-induced differentiation of 3T3-Ll fibroblasts (Sale et al., 1995). In another study, differentiation could be activated by v-raf, as scored by the accumulation of fat droplets and transcription of the aP2 lipid-binding protein, but activation of ERK or Rsk was not observed under these conditions,

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suggesting that differentiation through Ras and Raf occurs through pathways separate from those involving MKWERK (Porras et al., 1994). More recent studies have shown that the expression of constitutively active MKKl inhibits insulin-induced adipocyte differentiation, whereas the PD98059 MKK inhibitor suppresses ERK activation, with no effect on differentiation (Font de Mora et al., 1997). It thus appears that the primary effect of MKK/ERK is to negatively regulate adipogenesis and that v-raf may positively regulate differentiation through an alternative pathway. Among the important tissue-specific regulators of adipogenesis are the transcriptional activators CCAAT/enhancer-binding protein (C/EBPa, p, 6) and peroxisomal proliferator activated receptor y2 (PPAR-y); the latter mediates differentiation in response to thiazolidinediones and 15-deoxy-Af2>l4prostaglandin 52 (Wu, Z. et al., 1996). PPAR-y levels increase concomitantly with CEBPa, and together they synergize in promoting differentiation (Lin and Lane, 1992; Tontonoz et al., 1994; Brun et al., 1996). The inhibition of adipocyte differentiation by MKWERK can be explained by the finding that ERK2 phosphorylates PPAR-y2, most likely at Ser,,2. Mutation of Ser, 12 to Ala did not affect ligand-induced transcription, but relieved repression of transcription in response to phorbol ester (Hu et al., 1996), suggesting that ERK phosphorylation inhibits adipocyte differentiation by suppressing PPARq2 transactivation. The C/EBP family member, CHOP/ Gaddl53, inhibits adipocyte differentiation through heterodimerization with other C/EBP family members. In separate studies, phosphorylation of CHOP/Gaddl53 by p38 MAPK was found to be necessary for a full inhibitory effect (Wang and Ron, 1996), providing a mechanism by which ERK and p38 MAPK cooperate to antagonize differentiation in this system.

VI. YEAST MAPK PATHWAYS The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have provided an important complement to metazoan systems in the dissection of MAPK pathways. Through the use of genetic and molecular approaches, five distinct MAPK pathways have been revealed in S . cerevisiae and three in S. pombe. There are striking similarities between yeast and metazoan MAPK signaling, especially at the level of the MAPK module, phosphatase regulation, and transcriptional control. However, the yeasts lack homologs of Raf and appear to signal exclusively through MEKK-like kinases. In addition, the yeasts have thus far been shown to use heterotrimeric G protein-coupled seven transmembrane domain receptors and, in one case, a two-component system, thus receptor tyrosine kinases appear to be utilized exclusively in multicellular systems. Nevertheless, yeast models have

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led to important insights into the dual function of small GTPases in cytoskeletal organization and signaling, as well as mechanisms of heterotrimeric G protein signaling to MAPK modules.

A. Saccharornyces cerevisiae (Budding Yeast) 1. PHEROMONE RESPONSE PATHWAY

Mating in S . cerevisiae occurs between haploid cells of opposite mating types (aor a), and is characterized by the induction of mating-specific genes, cell cycle arrest, and cell morphology changes. This process occurs through activation of the pheromone response pathway, which employs a MAPK module composed of STE11, STE7, and FUS3/KSS1 (Fig. 2). The pheromone response is initiated when a or a peptide mating pheromone binds the appropriate seven transmembrane domain cell surface receptor (STE2 or STE3, respectively), which activates an associated heterotrimeric G protein through guanine nucleotide exchange, releasing Ga-GTP from GPY.The GPr dimer (STE4 and STE18) activates the downstream MAPK cascade through an incompletely understood mechanism, resulting in the activation of STE20 serinehhreonine kinase. The small GTPase, CDC42, and its GTP exchange factor, CDC24, are required in the pheromone response pathway. CDC42 binds to and activates STE20 (Simon et al., 1995; Zhao, Z. et al., 1995), as observed with mammalian Cdc42 and PAK. Although STE20 is required for mating, this interaction functions to localize STE20 to sites of bud emergence (Peter et al., 1996; Leberer et al., 1997) and may not be a necessary event in MAPK signaling. Once activated, STE20 phosphorylates and presumably activates STEll (MEKK) (Wu et al., 1995), although it is not clear how phosphorylation leads to activation. In analogy to the mammalian kinase cascades, STEll phosphorylates and activates STE7 (MKK) (Neiman and Herskowitz, 1994), which in turn phosphorylates and activates FUS3 and KSSl (MAPKs) (Gartner et al., 1992; Errede et al., 1993; Ma et al., 1995). FUS3 has the dual task of controlling the transcription of genes involved in mating and inducing cell cycle arrest. STE12, a transcription factor, and FAR1, a cell cycle regulator, are both substrates for FUS3 (Elion et al., 1993; Peter et al., 1993). Phosphorylation of STE12 is thought to activate transcription of mating specific genes, such as FUSland FARl (Trueheart et al., 1987; Oehlen et al., 1996), and phosphorylation of FARl arrests the cell cycle at G1 by associating with and inhibiting the cyclin-dependent protein kinase, CDC28-CLN2 (Peter et al., 1993; Tyers and Futcher, 1993; Peter and Herskowitz, 1994). Hence, as in mammalian systems, external signals received at the membrane are transduced to the nucleus via a MAPK cascade. STE5 is a signaling molecule that appears to serve as a scaffold for other

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components in the cascade (Choi et al., 1994; Printen and Sprague, 1994; Marcus et al., 1994). STE11, STE7, and FUS3/KSS1 all bind independently to different regions of STES, thus enabling association into a multikinase complex. Only the active form of STEl1 is present in the STES complex, suggesting a mechanism whereby STES binding facilitates STEl1 activation. Indeed, the N terminus of STEll (amino acids 1-435), which is required for STE5 interaction, also functions as a negative regulatory domain (Cairns et al., 1992). STES complexes also bind hypophosphorylated STE7, suggesting that STES may recruit active STEll to inactive STE7 and FUS3/KSS1. Activated STE7 and FUS3KSS1 may then be released, allowing FUS3/KSS1 to phosphorylate downstream targets (Choi et al., 1994). In absence of STES (ste5A),complexes among STE11, STE7, and FUS3KSS1 are still able to form, suggesting that other mechanisms exist to account for the role of STES in mating. However, although STEll and STE7 interact with FUS3/KSSl, STEll associates only marginally with STE7 (Choi et al., 1994; Printen and Sprague, 1994; Bardwell et al., 1996). Hence, facilitation of multikinase complexes by STES is probably important for signal transmission. Protein-protein interactions are implicated with other components that play a vital role in the pheromone response pathway. STE4 ( GP)binds to the N terminus of STES, potentially mediating signaling from heterotrimeric G proteins to the MAPK module (Whiteway et al., 1995). STE4 also binds the N terminus of the guanine nucleotide exchange factor CDC24 (Zhao, Z . et al., 1995).This suggests a model in which the STE4-STES complex activates CDC42 and recruits STE20 (via STE4), bringing it in proximity to its substrate, STEll (via STES). The SH3 domain-containing BEMl protein, involved in cell polarity, is required for efficient signaling in the pheromone response pathway. In a situation analogous to STE4, BEMl directly binds CDC24 (Peterson et al., 1994) and interacts with both STE20 and STES, as well as actin (Leeuw et al., 1995). In addition to binding the STES complex, BEMl associates with FARl (Lyons et al., 1996). Conceivably, BEMl could serve to localize the MAPK module, including its upstream activating components as well as the downstream FARl substrate, at the shmoo tip via its association with the actin cytoskeleton. The involvement of BEMl/CDC24/ CDC42 in the pheromone response pathway demonstrates that components necessary for cytoskeletal reorganization are also fundamental in signal transduction, reminiscent of Rac/Rho function in mammalian systems. In addition to facilitation of signaling through complex formation, STES might prevent cross-talk between the pheromone response pathway and the other S. cerevisiae MAPK pathways. In support of this, a STE7 gain of function mutant can complement a mutation in the cell wall integrity pathway (bcklA) only when it is overproduced or when STES is missing (Yashar et al., 1995). Desensitization of the pheromone response pathway involves dephosphory-

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lation and inactivation of FUS3. The dual specificity phosphatase MSGS (Doi et al., 1994) appears to regulate this process because loss of the MSGS gene results in diminished desensitization. Thus, a phosphatase homologous to the VHI-like class of dual specificity phosphatases, which in humans includes MKP-1 and PAC1 (Table I), is involved in inactivating yeast MAPK. Mating pheromone treatment enhances MSGS expression by fivefold within 30 min. This supports a negative feedback mechanism in which activation of FUS3 causes the transcriptional upregulation of MSGS, as is seen with mammalian MKP-1 and MKP-2 on serum stimulation (Brondello et al., 1997).

2. INVASIVE/PSEUDOHYPHAL GROWTH Invasive growth, characterized by filament formation and agar penetration, occurs in haploid yeast (aor a mating type) when nutritionally starved for nitrogen (Roberts and Fink, 1994). This pathway shares many components with the pheromone response pathway, including STE20 (PAK), STEl1 (MEKK), STE7 (MKK), and STE12 (transcription factor). Invasiveness does not require pheromone receptor, G proteins (STE4, STE18), STES, or FAR1, leaving the signal input component unknown. Deletion of both FUSS and KSSl does not impair invasive growth; however, loss of KSSl alone shows impaired agar penetration, suggesting that KSS 1 regulates the invasive response. Indeed, KSSl in its inactive state negatively regulates invasive growth, yet on activation by STE7, KSSl promotes invasiveness (Cook et al., 1997). Interestingly, mating specific genes are not induced, despite the presence of STE12 in the invasive growth pathway (see below). Two homologous proteins, DIGl and DIG2 (RST1 and RST2), have been identified that suppress invasive growth (Cook et al., 1996; Tedford et al., 1997). Both DIGl and DIG2 coprecipitate with KSSl and FUS3, indicating that they interact in v i m , and DIG112 bind STE12. DIGl colocalizes with KSSl in the nucleus, and both DIGs are phosphorylated by KSS1. These data suggest a model in which DIGs inhibit transcription from filamentous growth response elements (FREs), perhaps through binding of STE12. However, on pathway stimulation induced by starvation, KSSl might phosphorylate DIG112, thereby relieving transcriptional repression and allowing invasive growth. Pseudohyphal growth occurs in diploid ( d a ) cells and, like invasive growth, entails the formation of agar-penetrating filamentous growth in response to nitrogen starvation (Gimeno et al., 1992; Kron et al., 1994). STE20, STEl1, STE7, and STE12 are essential to pseudohyphal growth (Liu et al., 1993); and, as is the case for the invasive pathway, KSSl is the MAPK targeted by STE7 (Cook et al., 1997). Upstream components have also been further characterized in this system. Constitutive activation of U S 2 (through

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mutant RAS2""'19) enhances pseudohyphal growth (Gimeno et al., 1992). This may be mediated through CDC42, which has also been shown to regulate filamentous growth through binding of STE20, and functions downstream of RAS2 (Mosch et al., 1996; Peter et al., 1996; Leberer et al., 1997). However, RAS2 does not function in the pheromone response pathway and thus mediates signaling from a different source into the same MAPK module. Similarly, the 14-3-3 homologs BMHl and BMH2 are essential for pseudohyphal growth signaling, but not pheromone signaling (Robertset al., 1997). BMHl and BMH2 may function in the pathway via interaction with STE20. Activation of the pseudohyphal growth pathway does not activate mating specific genes, analogous to the invasive response (Mosch et al., 1996). Rather, STEl2 and the transcription factor TECl cooperatively bind to FREs to promote transcription required for both invasive and pseudohyphal development (Gavrias et al., 1996; Madhani and Fink, 1997). Other possible targets of the pseudohyphal growth MAPK module are MSSlO (a transcription factor) and MUCl (homologous to mucin-like membrane proteins), both of which are essential for pseudohyphal differentiation (Lambrechts et al., 1996).

3. HIGH OSMOLARITY RESPONSE (HOG PATHWAY) In S. cereuisiae, increases in external osmolarity are compensated for by increased glycerol synthesis and decreased glycerol permeability (Luyten et al., 1995). This high osmolarity glycerol response is rare among eukaryotic signaling pathways in that it combines a two-component response regulatory system (reviewed by Appleby et al., 1996) with a MAPK module composed of SSK2/SSK22 (redundant MEKKs), PBS2 (MKK), and HOGl(MAPK) (Fig. 2). Two distinct cell surface receptors, SLNl and SHOl, act as osmosensors that regulate the HOG cascade. SLNl was initially identified in a screen for synthetic lethal mutations requiring the ubiquitin protein-degradation pathway (Ota and Varshavsky, 1993). Both PBS2 and HOG1 were identified as osmoregulation-defective mutants (Brewster et al., 1993), and SSK2 and SSK22 were discovered in a screen for extragenic suppressor mutants of the lethal SLNl null mutation (Maeda et al., 1995). Strikingly, SLNl is a transmembrane protein with homology to bacterial two-component receptors, containing osmotic sensor, histidine kinase transmitter, and phosphate receiver domains. The remainder of this two-component system includes the response regulators YPDl and SSKl (Maeda etal., 1994; Posas et al., 1996), activating the HOG pathway at the level of SSK2 and SSK22 (Maeda et al., 1995). Under conditions of low osmolarity, SLNl autophosphorylates at His,,, in its transmitter domain. The phosphoryl group is first transferred to Asp,,,, in the SLNl receiver domain, from which it is transferred to His,, on YPD1. Finally, the

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phosphoryl group is passed to Asp,,, in the C-terminal receiver domain of SSKl (Posas et al., 1996).SSKl is phosphorylated and inactive when the external osmolarity is low. Under conditions of high osmolarity, the SLNl histidine kinase is inactivated and the resulting unphosphorylated SSKl activates the downstream SSK2/SSK22, most likely by binding the N-terminal inhibitory domain of SSK2 or SSK22 and relieving autoinhibitory constraints (Maeda et al., 1994). Upon activation, SSK2 or SSK22 phosphorylates and activates PBS2, which in turn phosphorylates and activates H O G l . Thus, histidine kinase activity negatively regulates HOG1, and slnld mutant strains are lethal due to hyperactivation of the HOG pathway. The second osmosensor, SHOl, is a putative transmembrane protein needed for viability in slnld strains. SHOl associates with PBS2 through the binding of its SH3 domain to a proline-rich domain in PBS2 (Maeda et al., 1995). This interaction activates PBS2 through phosphorylation; however, neither SSK2 nor SSK22 is involved. Instead, SHOl binding recruits PBS2 to a multikinase complex with STEll and HOG1, although no crossregulation of HOG by pheromone is apparent (Posas and Saito, 1997). Once activated, H O G l completes the signaling response by inducing the transcription of genes necessary for maintaining osmotic balance. Among these are glycerol-3-phosphate dehydrogenase (GPD1) (Albertyn et al., 1994) and glycerol-3-phosphatase (GPP2) (Norbeck et al., 1996), both essential for glycerol synthesis. Other HOG1-regulated genes include a cytosolic catalase T, CTTl (Schuller et al., 1994), and a small heat shock protein, HSP12 (Varela et al., 1995), both of which are under the control of stress response elements (STREs). The zinc finger transcription factors MSN2 and MSN4 are important regulators of STRE-dependent promoters and are required for activation of C TTl and HSP12 genes (Schmitt and McEntee, 1996; Martinez-Pastor et al., 1996). Strains deficient in MSN2 and MSN4 are less sensitive to high osmolarity than hogld strains but are still viable; therefore, not only do these transcription factors function in HOG signaling, other factors are likely to be involved. The HOG pathway can be downregulated by protein tyrosine phosphatases PTP2 (Ota and Varshavsky, 1992; Guan et al., 1992; James et al., 1992) and PTP3 (Jacoby et al., 1997; Wurgler-Murphy et al., 1997), as well as PTC1, PTC2, and PTC3, which are homologous to the mammalian serinehhreonine phosphatase, PP2C (Maeda et al., 1993; I. Ota, personal communication). PTPs 2 and 3 and PTCs 1-3 are able to suppress lethality in slnld strains when overexpressed, indicating that these phosphatases suppress hyperactivation of the HOG pathway (Maeda et al., 1994; I. Ota, personal communication). PTP2 and PTP3 target HOG1, whereas the substrate specificities of PTCs 1, 2, and 3 have yet to be determined in this system (Table I). It has been suggested that the PTCs regulate both PBS2 and H O G l activity, although this has not been directly demonstrated. Both

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PTP2 and PTP3 are transcriptionally induced in response to hyperosmolarity in a HOG1-dependent manner (Jacoby et al., 1997), thus downregulation of the pathway can be controlled through a negative feedback mechanism. 4. CELL WALL INTEGRITY (PKC PATHWAY)

A MAPK cascade modulates part of a pathway regulating cell wall integrity. S. cerevisiae has a single PKC gene (PKC1) that is closely related to the mammalian PKC a, p, and y isoforms (Levin et al., 1990). Mutations that inactivate PKCl result in cell lysis, caused by deficiencies in cell wall construction (Levin and Bartlett-Heubusch, 1992; Paravicini et al., 1992). Downstream of PKCl is an MEKK (BCKUSLKl), which, when mutated, also results in cell lysis (Lee and Levin, 1992; Costigan et al., 1992). BCKl has a PKC consensus phosphorylation site at Ser,134, and PKCl selectively phosphorylates BCKl in vitro (Levin et al., 1994), suggesting that PKCl activates BCKl through phosphorylation. In addition, two functionally redundant MKKs in this pathway (MKK1 and MKK2) suppress a BCKl deletion mutant when overexpressed (Irie et al., 1993). MKKl and MKK2 are upstream of and activate a MAPK, MPKUSLT2 (Torres et al., 1991; Lee et al., 1993), which, like FUS3, is negatively regulated by the dual specificity phosphatase MSG5 (Watanabe et al., 1995). Although the PKCl pathway does not appear to involve a STE5-like protein, MKKl interacts with PKC1, whereas MKKl and MKK2 bind both BCKl and Mpkl (Soler et al., 1995; Paravicini and Friedli, 1996). Thus, the kinase components form a multienzyme complex through association with MKK1/2. Although each component in this pathway (BCK1, MKK1/2, and MPK1) yields a cell lysis defect when deleted, none is as severe as the loss of PKC1. This implies that the MAPK module functions only in one branch of a bifurcated pathway regulated by PKCl (Errede and Levin, 1993). Upstream regulation of PKCl involves a number of stimulatory inputs. Tyrosine phosphorylation and activation of MPKl increase in response to hypotonic shock (Davenport et al., 1995) or mild heat shock (Kamada et al., 1995). In addition, MPKl is activated during periods of polarized cell growth (budding) in a CDC28-dependent manner (Mazzoni et al., 1993; Zarzov et al., 1996; Marini et al., 1996). Each of these events disrupts the structural integrity of the plasma membrane, suggesting a mechanism whereby plasma membrane stretch activates mechanosensitive ion channels, resulting in an influx of Ca2+(Kamada et al., 1995). Increased intracellular Ca2+ might activate PKCl directly or indirectly through the stimulation of phospholipase C (PLC) and diacylglycerol synthesis. In the case of polarized cell growth, CDC28 upregulates diacylglycerol production, possibly through activation of a phosphatidylcholine-

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specific PLC (Marini et al., 1996). Although PKCl is not activated by phospholipids, Ca2+,or DAG in uitro, it is conceivable that the proper conditions have not been found (Watanabe etal., 1994).For example, the phospholipiddependent regulation of PKCl is facilitated by RHOl-GTP, a homolog of mammalian RhoA, which binds PKCl at its pseudosubstrate site and C1 domain (Nonaka et al., 1995). When complexed with RHO1-GTP, PKCl is strongly activated by phosphatidylserine (Kamada et al., 1996),which serves as the exclusive cofactor in the R H O l P K C l complex, as neither Ca2+ nor DAG is stimulatory. A R H O l loss of function mutation is complemented by overexpression of MPKl or a gain of function MKK1, and R H O l is required for MPKl activation during heat shock (Kamada et al., 1996).These results suggest that PKCl is a target for RHOl and that RHO1-dependent stimulation of PKCl activates the downstream MAPK cascade (Nonaka et al., 1995). Other evidence suggests that PKCl might be regulated by phosphoinositides. A yeast phosphatidylinositol4-kinase (STT4) has been shown genetically to function upstream of PKCl and BCKl (Yoshida et al., 1994). However, a strain bearing an STT4 deletion is incompletely suppressed by overexpression of PKCl or a gain of function BCKl mutant, implying that a second branch point likely exists between STT4 and PKCl . Targets of the MAPK cascade in the PKCl pathway are just beginning to be defined. Two high-mobility group (HMG)-like proteins (NHP6A and NHP6B) function downstream of MPK1, although they are not likely to be direct targets (Costigan et al., 1994).A transcription factor (RLM1)with homology to the MADS box family is a probable target of MPKl induction (Watanabe et al., 1995). Five genes involved in cell wall biosynthesis are upregulated by MPKl, including those encoding enzymes needed in the synthesis of (1-3)-P-glucan, (1-6)-P-glucan, chitin, and N- and 0-linked mannoproteins (Igual et al., 1996).Upstream regulatory promoter sequences and MPK1-specific transcription factors that regulate these genes have yet to be determined.

5. SPORE FORMATION Sporulation occurs in diploid yeast during conditions of nitrogen starvation in the presence of a nonfermentable carbon source. A MAPK gene, SMK1, has been identified as a regulator of spore formation (Krisak et al., 1994). A STE20-like serinekhreonine protein kinase, called SPS1, is also required in sporulation (Friesen et al., 1994). Yeast strains mutant in either of these genes are defective in spore wall biosynthesis. Both SMKl and SPSl transcripts are expressed in a sporulation-specific manner and are required for the normal expression of late sporulation-specific genes. SPSl and SMKl probably represent components in a MAPK module specific to the sporula-

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tion process. However, the homologs of MEKK and MKK in this pathway have not yet been identified.

B. Schizosaccharomyces pom6e (Fission Yeast) 1. PHEROMONE RESPONSE PATHWAY

Sexual differentiation in S. pombe is similar in many respects to S. cuevisiae; however, there are fundamental disparities. In particular, fission yeast require a mating pheromone as well as nutritional starvation in order to mate. These are respectively mediated by the pheromone response pathway and the stress response MAPK pathway (discussed later). Hence, sexual differentiation involves two distinct MAPK signaling pathways. Another difference is that G protein signaling is transmitted through the Ga subunit in S. pombe. Furthermore, Ras is required for the pheromone response, whereas it has no such role in S. cerevisiae. Mating in S. pombe occurs between cells of opposite mating type (P or M). Initiation of the pheromone response takes place when P or M pheromones associate with their receptors (Mam2 or Mam3, respectively), stimulating the release of GTP-bound Ga. The Ga subunit, encoded by the gpal gene, is required for both mating and sporulation (Obara et al., 1991), although effectors that interact with GOiare not known at present. An essential constituent of the pheromone pathway is Rasl (Nadin-Davis et al., 1986),which regulates a MAPK module composed of Byr2 (MEKK), Byrl (MKK), and Spkl (MAPK) (Nadin-Davis and Nasim, 1988, 1990; Wang et al., 1991; Toda et al., 1991). Each component of this MAPK cascade is necessary for the execution of sexual differentiation. Genetic evidence indicates that Ga and Rasl coordinately stimulate the kinase module, converging at B y d (Xu et al., 1994). Rasl directly binds to the N terminus of Byr2 in a GTP-dependent manner, although this interaction has not been reported to activate Byr2 (Van Aelst et af., 1993; Masuda et al., 1995). Rasl also indirectly activates the MAPK module through the STE20 homolog, Shklmakl. Two-hybrid interaction experiments have shown that Rasl-GTP forms a complex with Scdl (CDC24 homolog), Scd2 (BEM1 homolog), and the small GTPase Cdc42, important for both mating and morphogenesis (Chang et al., 1994). Shkl binds to Cdc42 both in vitro and in vivo, and thus is likely to be a Cdc42 effector (Marcus et al., 1995; Ottilie et al., 1995). Shkl restores mating in ste20A strains of S. cerevisiae, and recombinant Shkl is able to activate both ERK2 and JNUSAPK in Xenoptrs oocyte extracts (Polverino et al., 1995),supporting its involvement in MAPK signaling. Another protein essential for sexual differentiation, Ste4, binds the N-terminal regulatory domain of Byr2 at a site distinct from the Ras-1 bind+

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ing domain (Okazaki et al., 1991; Barr et al., 1996). Ste4 acts upstream of Byr2, and Byr2 requires Ste4 binding for complete function. Therefore, full activation of the MAPK cascade involves multiple inputs at the point of Byr2. Signaling downstream of Byr2 is conducted through the Byr2-Byrl-Spkl pathway, which is structurally and functionally homologous to the S. cerevzsiae STEl lSTE7-FUS3KSSl module (Neiman et al., 1993). Consistent with homology and complementation data, tyrosine phosphorylation of Spkl is dependent on Byrl (Gotoh et al., 1993).The matl-Pm gene, which controls entry into meiosis, is positively regulated by signaling through the pheromone response pathway (Aono etal., 1994).The transcription factor, Stell, is a regulator of numerous mating-specificgenes, including matl-Pm; however, it has not been shown to be a substrate of Spkl (Sugimoto et al., 1991).

2. STRESSEEXUAL RESPONSE PATHWAY In S. pombe, multiple forms of environmental stress activate a MAPK cascade with components Wikl/Wakl/Wis4 (MEKK), Wisl (MKK) and Spcl/ Styl/Phhl (MAPK) (Warbrick and Fantes, 1991; Millar et al., 1995; Shiozaki and Russell, 1995a; Kato et al., 1996; Samejima et al., 1997; Shieh et al., 1997; Shiozaki et al., 1997). Spcl is phosphorylated and activated by Wisl in response to hyperosmolarity, starvation, oxidative stress, and heat shock (Millar etal., 1995; Shiozaki and Russell, 1995a; Degols et al., 1996). Like the HOG pathway, the Wikl-Wisl-Spcl module is regulated by an upstream two-component system (Shieh et al., 1997). Mcs4 is homologous to S. cerevisiae SSKl and functions upstream of Wikl, which activates Wisl by phosphorylation. Wisl can also be activated by Win1 in response to osmotic stress independent of Mcs4/Wikl, while Spcl is activated by heat and oxidative stress independent of Wisl, perhaps involving Pypl phosphatase inactivation (Samejima et al., 1997). This MAPK pathway is regulated by several phosphatases from both the PTPs and PP2C families, in a situation analogous to the HOG pathway. The PTPs are Pypl and Pyp2, which are negative regulators of mitosis (Ottilie et al., 1991, 1992; Millar et al., 1992; Hannig et al., 1994). Pypl and Pyp2 both form complexes with Spcl and efficiently inactivate it by direct dephosphorylation of Tyr173 (Shiozaki and Russell, 1995a; Millar et al., 1995). Interestingly, Spcl activation induces expression of Pyp2, forming a negative feedback loop (Millar et al., 1995; Degols et al., 1996; Wilkinson et al., 1996). The PP2C-related phosphatases, Ptcl, Ptc2, and Ptc3, are also implicated in downregulating the Wisl/Spcl pathway, and mutants in both Spcl and Wisl suppress the lethality of a AptclAptc3 double mutant (Shiozaki et al., 1994; Shiozaki and Russell, 1995b). However, the particular substrate(s) for Ptc has yet to be determined. Dual specificity phosphatases that act on this pathway have not been reported at this time.

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Activation of Spcl is required for the transcription of genes involved in stress responses and sexual development. Examples of stress-responsive genes induced by the Spcl pathway are glycerol-3-phosphate dehydrogenase (gpdl+),trehalose-6-phosphate synthase (tpsl+),and catalase (A) (Aiba et al., 1995; Degols et al., 1996; Shiozaki and Russell, 1996; Wilkinson et al., 1996). Spcl is also essential for expression of the Stel 1transcription factor, which regulates sexual differentiation genes in response to nitrogen starvation (Kato et al., 1996; Shiozaki and Russell, 1996). This implicates the Wisl/Spcl pathway as an essential element in sexual differentiation. Atfl, a transcription factor homologous to mammalian ATF-2 (Takeda et al., 1995), is a likely effector of transcriptional regulation by Spcl. In vitro, Atfl and Spcl physically associate and Spcl phosphorylates Atfl . Furthermore, stress-induced expression of gpdl+, tpsl+, catalase, pyp2+, and stel 1 requires Atfl (Shiozaki and Russell, 1996; Wilkinson et al., 1996). However, Atfl deletion mutants are not defective in mitosis, indicating a branch point in the Wisl/Spcl pathway between mitotic and stress responses. In S. pombe, the stress response pathway may indirectly stimulate the pheromone response pathway, an example of cross-regulation between MAPK cascades. Transcription of the Byr2 regulator, Ste4, is dependent on Stell (Okazaki et al., 1991). Thus, regulation of the pheromone response pathway through Ste4 may occur following Stel 1 expression in response to the stress pathway. This may explain the dual requirement for both pheromone factor and nutritional starvation in the mating response. +

3. CELL WALL INTEGRITY PATHWAY A third MAPK isolated in S . pombe, called Pmklhmpl, is a potential regulator of cell wall integrity (Toda et al., 1996; Zaitsevskaya-Carter and Cooper, 1997). p m k l d strains have multiple phenotypes including cell wall weakness, unusual cell shape, defective cytokinesis, and altered cation sensitivity. Pmkl shares 65% identity with S. cerevisiae Mpkl, and Mpkl overexpression is able to rescue p m k l d phenotypes. However, despite the similarities between these two MAPKs, Pmkl is not activated by either hypoosmolarity or heat shock nor does it appear as though Pmkl regulates cell wall integrity in a linear pathway with PKC. S . pombe has two known PKCs, p c k l + and pck2+ (Mazzei et al., 1993; Toda et al., 1993), encoding homologs of the 6, E, and q isoforms of mammalian PKC. Strains lacking Pck2 have a cell wall defect (Shiozaki and Russell, 1995b), that is more severe than the Pmkl null strain. However, genetic data argue that Pmkl and Pckl/2 do not function independently in the regulation of cell wall integrity, but rather in a coordinate manner. One possibility is that Pckl/2 regulate a bifurcated pathway with Pmkl functioning in one of the branches.

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VII. INTRACELLULAR TARGETING AND SPATIAL REGULATION OF MAPK PATHWAY COMPONENTS A. Signaling Complexes An important principle that has emerged from studies on kinase cascades is that protein kinases and their targets interact in a way that confers specificity in signaling. Part of this specificity can be attributed to recognition motifs within the active site of each protein kinases. For example, a systematic comparison of different chimeras between ERK2 and p38 MAPK showed that helix C within the N-terminal lobe of the conserved kinase structure is an important determinant for recognition by growth factor vs stress signaling (Brunet and PouyssCgur, 1996). Presumably, this domain forms part of the recognition surface between MKKs and MAPKs, as observed in the recognition surface between cyclin A and cdk2 (Jeffrey et al., 1995). Domains outside the active site may extend the interaction surface between various enzymes and their substrates. For example, stable complexes between STE7 and FUS3 involve interactions between FUS3 and the STE7 N terminus (Bardwell et al., 1996), and a proline-rich domain between subdomains 9 and 10 of MKKl is important for association between MKKl and Raf-1 (Papin et al., 1996). Extensive regulatory domains containing conserved interaction motifs are present within several protein kinases, such as Raf-1, MEKKs, PAKs, and multilineage kinases, providing mechanisms to confer multiple selective interactions that augment active site recognition. Complexes formed through such extensive protein-protein interactions would be expected to facilitate signal transmission and quite possibly provide an additional insulating function, preventing cross-regulation between homologous enzymes in growtWdifferentiation vs stress regulatory pathways. The best example for this is in S. cerevisiae, where complex formation of STE5 with STE4, BEM1, STE11, STE7, and/or FUS3 provides a mechanism to facilitate the interaction between several pathway components. However, STE5 appears to be unique to the pheromone response pathway. Homologs have not been found in other systems, and STES does not interact with S. pornbe MEKK or MKK (Byr2, Byrl) nor does it bind to mammalian MEKK, MKK, or Raf (Marcus et al., 1994).Thus, in other cell types, conserved binding motifs within the kinases themselves might provide the scaffolding for interactions that facilitate signaling. Most of the evidence for stable complexes formed between protein kinases and their substrates is derived from biochemical coprecipitation experiments as well as from two-hybrid interaction screens. Raf-1 and B-Raf form stable complexes with MKKl and ERKs, mediating the interaction of these kinases with Ras (Moodie et al., 1993; Jelinek et al., 1996; Papin et al., 1995, 1996) and potential interactions with other proteins such as 14-3-3 or KSR.

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Raf-1 also interacts with GPr subunits and tyrosine kinases Src and Fyn (Cleghon et al., 1994; Pumiglia et al., 1995). Evidence also exists for stable interactions between Ras with MEKK (Russell et al., 1995), Rac-GTP or Cdc42-GTP with PAK20 or MLK-3 (Bagrodia et al., 1995; Teramoto et al., 1996), MLK-3 with JNKUSAPKl or MKK3 (Tibbles et al., 1996), and JNKWSAPKl with JNWSAPK (Zanke et al., 1996). Interactions between MAPKs and their downstream substrates have also been detected biochemically, best exemplified by JNWSAPK interactions with c-Jun, which are stable enough to enable the detection and purification of JNWSAPK (Hibi et al., 1993). These interactions are mediated through interactions between a JNK docking site removed from the phosphorylation site on c-Jun and a specificity determination site on JNK/SAPK outside the kinase catalytic cleft (Kallunki et al., 1994,1996). Other substrates that interact with JNWSAPK include transcription factors ATF and Elk-1 (Bocco et al., 1996; Gupta et al., 1996), and p38 MAPK has been found complexed with MAPKAP kinase 2 and 3pk (McLaughlin et al., 1996; Krump et al., 1997). Substrates that copurify with ERK include MAPKAP kinase-1, cMyc, and c-Jun (Scimeca et al., 1992; Hsiao et al., 1994; Gupta and Davis, 1994; Bernstein et al., 1994).

B. Nuclear Translocation of MAPK and MKK Signaling from an extracellular ligand at the plasma membrane requires the migration of at least one of the components in the signaling cascade into the nucleus, where an important end result is transcriptional activation. In some cases, MAPKs fulfill this function, enabling regulation of gene expression through phosphorylation of nuclear transcription factors. The localization of ERKl and ERK2 is predominantly cytoplasmic in quiescent cells (Chen, R. et al., 1992; Lenormand et al., 1993). However, upon serum or growth factor stimulation, a fraction of cytoplasmic ERK translocates into nuclei (Chen, R. et al., 1992; Traverse et al., 1992; Lenormand et al., 1993; Gonzalez et al., 1993). Translocation occurs rapidly (within 5-30 min, depending on cell type) and typically endures for several hours. Similarly, JNKUSAPK is both cytoplasmic and nuclear in unstimulated cells, but upon UV irradiation migrates into the nucleus (Cavigelli et al., 1995). Although y radiation also stimulates JNKUSAPK activity, it does not appear to cause nuclear translocation (Chen, Y. et al., 1996a).Although a study of endogenous p38 MAPK translocation has yet to be reported, overexpressed p38 MAPK localizes at the cell periphery, in the cytoplasm, and in the nucleus; however, UV irradiation does not lead to its redistribution between cytoplasm and nucleus (Raingeaud et al., 1995). ERK3, unlike the other MAPKs, is constitutively nuclear (Cheng et al., 1996).

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Mechanisms for MAPK translocation have yet to be determined. Neither ERKl nor ERK2 contain nuclear localization or nuclear export signals, and ERKs are below the size limit for passive diffusion through nuclear pores, suggesting that nuclear export may be passive. One possibility is that ERK is retained in the cytoplasm through its interaction with a cytoplasmic localization factor. One candidate for this factor is MKK1, based on data showing a specific interaction of ERK with the N terminus of MKK, cytoplasmic retention of ERK upon expression of MKK, and reduced coimmunoprecipitation of MKK and ERK on cell stimulation (Fukuda et al., 1997). Interestingly, ERK nuclear translocation does not require its activation as nonphosphorylatable mutants ERK-Thr,,,Ala, Tyr,,,Phe or catalytically inactive ERK still translocate in response to serum (Gonzalez et al., 1993; Lenorrnand et al., 1993). However, the rapid uptake of ERK and its nuclear retention against a gradient following stimulation suggest that active import is also likely. Unlike the ERKs, MKKl and MKK2 are cytoplasmic before and after growth factor stimulation (Zheng and Guan, 1994b; Moriguchi et al., 1995a). MKK has a short N-terminal, leucine-rich stretch of amino acids (amino acids 32-44) that acts as a strong and autonomous nuclear export signal (Fukuda et al., 1996). Ovalbumin, covalently coupled to a synthetic peptide containing the MKK export sequence, is directed out of the nucleus immediately after microinjection. Thus, a mechanism is in place to export MKK to the cytoplasm, implying that nuclear import of wild-type MKK may occur under physiological conditions. One study has demonstrated serumdependent nuclear uptake of MKK mutants lacking the export sequence (Jaaro et al., 1997). This raises the question of whether MKK import is required for signaling. For example, MKK and ERK activation may be elevated over several hours following cell stimulation under certain conditions. The existence of high levels of nuclear phosphatases, including dual specificity phosphatases, which target ERK, necessitates a mechanism for maintaining elevated ERK activity in nuclei. Therefore, one possibility is that MKK nuclear uptake may serve as a means for maintaining active nuclear ERK.

VIII. FUTURE DIRECTIONS Eukaryotic MAP kinase cascades provide excellent examples of signal transduction mechanisms that embody key principles common to many, if not all, signaling pathways. These include regulation of reversible posttranslational modification by phosphorylation, sequential activation of multiple signaling components, and involvement of second messengers. Studies

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have revealed additional features of these pathways important for signal transmission, such as stable protein complex formation, translocation between intracellular organelles, cross-regulation, and the involvement of homologous kinase modules in parallel pathways. Many fundamental questions remain for future studies to investigate the mechanisms by which these pathways are regulated as well as the cellular responses that they control. For example, in several cases, the identification of pathway components is incomplete; in particular, many enzymes that mediate the activation of stress-activated kinases by upstream effectors, as well as the sensors for these stress signals, have yet to be identified. We also have little understanding of how these kinase cascades can control such diverse processes as cell transformation, stress responses, and cell differentiation, and why different types of cells respond to the same pathway in different ways. The substrates found so far for MAPKs are likely to be a small fraction of the total number of targets that exist. We have few clear examples of how phosphorylation regulates their function, and we have yet to identify other substrates for upstream kinases that enable these pathways to bifurcate. Finally, little is known of how the events in signaling and cross-regulation are coordinated in order to modulate and tune the cellular responses to these pathways. These kinase cascades are clearly not fabricated from assorted enzymes jumbled together, but require processes of ordered assembly and hierarchal activation to enable the efficient facilitation of signal transduction. The combined use of genetics, molecular biology, and biochemical approaches, together with newly acquired genomic sequences, now set the stage for further exciting research into this highly complex problem.

ACKNOWLEDGMENTS This work was supported by Grants GM48521 (N.G.A.)and GM18151 (P.S.S.) from the National Institutes of Health. We thank Katheryn Resing, Irene Ota, Loree Kim, and Donna Louie for many helpful discussions.

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FHIT in Human Cancer Cabdella Sozzi,"Kay Huebner,f and Carlo M. Crocet *Division of Experimental Oncology A, lstituto Nazionale Tumori, 201 33 Milan, Italy, and tKimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 191 07

I. Introduction A. Chromosomal Rearrangements and Loss of Heterozygosity of the Short Arm of Chromosome 3 in Tumors B. Fragile Sites and FRA3B 11. Cloning and Structural Features of the FHIT Gene 111. The Fhit Protein and Its Biochemical Properties IV. FHIT Abnormalities in Human Cancer A. Tumors of the Gastrointestinal Tract B. Tumors of the Aerodigestive Tract C. Tumors and Preinvasive Lesions of the Breast D. Other Tumors V. Conclusions and Perspectives References

1. INTRODUCTION

A. Chromosomal Rearrangements and Loss

of Heterozygosity of the Short Arm of Chromosome 3 in Tumors Chromosomal deletions and loss of heterozygosity (LOH) involving the short arm of chromosome 3 (3p)occur frequently in carcinomas of the lung, head and neck, kidney, breast, and other epithelial neoplasms (Devilee et al., 1989; Hibi et al., 1992; Lothe et al., 1989; Ogawa et al., 1991; Yang-Feng et al., 1993; Kohno et al., 1993; Maestro et al., 1993). However, the pattern of 3p losses in tumors is complex and involves several distinct regions: 3p12, 3 ~ 1 4 ~ 3 ~and 2 1 3p24-25. , Deletions of 3p are probably early events in cancerogenesis as they have been reported in preneoplastic lesions of the lung (Sozzi et a!., 1991; Sundaresan et al., 1992; Hung et al., 1995a), in benign proliferative breast diseases (Teixeira et al., 1996; Panagopoulos et al., 1996), and in oral leucoplakia (Ma0 et al., 199613; Roz et al., 1996). These findings, together with the identification of homozygous deletions in cancer cell lines, have led to the hypothesis that these regions are the sites of tumor Advances in CANCER RESEARCH 0065-230W98 $2S.00

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suppressor genes (TSG),whose inactivation is achieved by the loss of one allele and the presence of inactivating mutations or lack of expression in the remaining allele. These genetic changes ultimately result in the loss of the wild-type protein expression or function. Tumor suppressor activity has been demonstrated for the entire chromosome 3 in lung adenocarcinoma (Satoh et al., 1993), renal cell carcinoma (Shimizu et al., 1990; Yoshida et al., 1994; Sanchez et al., 1994), ovarian carcinoma (Rimessi et al., 1994), and mouse fibrosarcoma (Killary et al., 1992).The tumor suppressor activity was subsequently associated with portions of 3p, indicating the existence of two regions displaying such an activity. The region 3p22-p21 suppressed the tumorigenic properties of a mouse fibrosarcoma cell line (Killary et al., 1992), and the introduction of region 3~14-12into renal carcinoma cells with a translocation t(3;8) (p14.2;q24) resulted in partial suppression of tumor growth in nude mice (Sanchez et al., 1994). The region within 3p22-p21 has been narrowed down to an 80-kb clone from 31321.3 as demonstrated by suppression of tumor growth in vivo (Todd et al., 1996). However, no solid candidate for the tumor suppressor gene(s) in these regions has yet been identified. So far the only definite 3p-linked TSG is the Von Hippel-Lindau gene (VHL),located at 3p25. Von Hippel-Lindau disease is a familial cancer syndrome, dominantly inherited, that predisposes affected individuals to a variety of tumors, among them renal cell carcinomas. The VHL gene was shown to be mutated in the germline DNA of VHL disease families (Latif et al., 1993). The VHL gene is also inactivated in a considerable fraction of sporadic renal cancer (Gnarra et al., 1994) but is only rarely mutated in other tumors, such as lung lesions (Sekido et al., 1994). Region 3p14 is frequently lost in primary carcinomas (Rabbitts et al., 1989; Brauch et al., 1990; Hibi et al., 1992; Lubinski et al., 1994; Hung et al., 1995b; Druck et al., 1995; Pandis et al., 1996; Teixeira et al., 1996; Mao et al., 1996b; Roz et al., 1996) and is the site of homozygous deletions in a range of cancer-derived cell lines (Lisitsyn and Wigler, 1995; Kastury et al., 1996; Boldog et al., 1997). It also contains the 3p break of a hereditary renal-carcinoma-associated translocation, t(3;8) (p14.2;q24), which has been shown to segregate in a family with an early onset of bilateral and multifocal clear cell renal carcinoma (Cohen et al., 1979). FRA3B, another cytogenetic landmark in chromosome region 3 ~ 1 4 . 2 ,is the most active of the common constitutive aphidicolin-inducible fragile sites in the human genome (Glover et al., 1984).

B. Fragile Sites and FRA3B Fragile sites are specific regions of chromosomes that reveal cytogenetically detectable breaks when cells are exposed to certain chemical reagents or cul-

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ture conditions and are divided into two classes, common and rare (for review see Sutherland, 1991). Whereas common fragile sites are expressed in all individuals, although at different levels, rare fragile sites are present in a small proportion of the population and are heritable. Several folate-sensitive, heritable, X-linked, and autosomal fragile sites have been localized to unstable CCG or CGG repeats (Yu et al., 1991; Fu et al., 1991; Kremer et al., 1991) and were shown to be implicated directly in human diseases. The best known examples are the fragile X syndrome (FRAXA) and the X-linked mild mental retardation locus (FRAXE)characterized by the dramatically increased size of simple trinucleotide repeats, (CCG)n. This amplification of repeats is associated with abnormal methylation of the adjacent CpG islands and loss of expression of the FMRl and FMR2 genes (Verkerk et al., 1991; Pieretti et al., 1991; Knight et al., 1993; Mulley et al., 1995). A direct link between a fragile site and “in vivo” chromosome breakage has been demonstrated with the association between a chromosome deletion syndrome and a fragile site (Jones et al., 1995). In fact, the fragile site FRAllB at l l q 2 3 has been localized to the (CCG)n repeat of the CBL2 protooncogene. Jacobsen syndrome, recognized by specific dysmorphic features and moderate mental retardation, is characterized by a deletion of the long arm of chromosome 11 (llq23-qter). A proportion of Jacobsen syndrome patients have been shown to inherit a chromosome carrying a CBL2 expansion, which was truncated close to FRAllB. In addition, a CpG island adjacent to FRAllB was found to be methylated when its length exceeded certain limits. The rare dystamycin A-sensitive fragile site, FRAl6B located at 16q22.1, has also been isolated by positional cloning and is an expanded 33 bp ATrich minisatellite repeat (Yu et al., 1997). Therefore, the unstable repeat sequence mutation, so far associated with the increase in copy number of trinucleotide repeats, as mentioned earlier, is also a property of repeats of various compositions and also with various lengths of repeat motif. Common fragile sites represent a cytogenetic puzzle. The biologic role, if any, and the molecular basis for chromosomal fragility at common fragile sites are not known. The apparent association among common fragile site localization, cancer breakpoints, and genes involved in tumorigenesis (Yunis and Soreng, 1984; Le Beau, 1986) led to the hypothesis that fragile site breakage was involved in the chromosomal rearrangements and allele losses observed in malignant diseases. Common fragile sites are highly conserved during evolution (Schmid et al., 1985), are induced by agents that perturb DNA replication, are located in the euchromatic and late replicating genome regions, and are induced by agents known to attack chromatin DNase I hypersensitive sites, i.e., sequences associated with expressed genes (Yunis et al., 1987; Musio and Sbrana, 1996). In fact, common fragile site Xp22.1 is expressed only on the active X chromosome. Thus, common fragile sites could be associated with transcriptional activity. Consequently, the potential role of a fragile site located at the 5’ end of a tumor suppressor gene could

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be the loss of transcription of such a hypothetical gene as a result of fragile site expression. It is of interest that the rate of expression of common fragile sites can vary from person to person; occasionally, individuals are encountered who have high levels of expression of one of these fragile sites (Sutherland and Richards, 1995). The most active of the inducible common fragile sites of the human genome is FRA3B, contained in the 3p14.2 chromosomal band. FRA3B expression is observed after exposure of cultured cells to diverse mutagens and carcinogens, including benzo[a]pyrene diol epoxide, the ultimate carcinogen of benzo[a]pyrene (a major constituent of tobacco smoke) (Yunis et al., 1987) and ethanol (Kuwano and Kajii, 1987). A significantly increased frequency of FRA3B expression has been reported in peripheral lymphocytes of smokers (Kao-Shan et al., 1987). Aphidicolin induction of breakage and rearrangement of FRA3B in somatic cell hybrids resulted in the generation of hybrid clones with human/ hamster translocations involving breakpoints at FRA3B (Glover and Stein, 1988; Paradee et al., 1995). Subsequent studies of the position of hybrid clone breakpoints (Wilke et al., 1994; Boldog et al., 1994) have shown that the genomic region involved was up to 100 kb, suggesting that FRA3B may represent a region of fragility rather than a single site. It has been shown that an area of frequent breaks within FRA3B coincides with the spontaneous HPV16 integration site, offering direct evidence for the coincidence of viral integration sites and fragile sites (Wilke et al., 1996).

11. CLONING AND STRUCTURAL FEATURES

OF THE FHIT GENE The observations summarized in Section I,A indicate that the 3p14.2 region probably harbors one of the long sought tumor suppressor genes on the short arm of chromosome 3. The isolation of a YAC contig covering the t(3;8) break and FRA3B (Wilke et al., 1994; Boldog et al., 1994; Michaelis et af., 1995; Kastury et al., 1996) and the development of STS markers allowed the definition of homozygous deletions in a range of cancer-derived cell lines (Kastury et al., 1996). Ohta et al. (1996) developed a cosmid contig covering the common homozygously deleted region and identified and characterized a gene that is partially deleted in uncultured tumors of the aerodigestive tract and other organs. This gene has been designated the fragile histidine triad gene or the FHZT gene. The cosmid contig was assembled from cosmids subcloned from the library prepared from YAC 648D4, the shorter YAC clone covering the commonly deleted region (Fig. 1).Individual cosmids were used in exon-trapping

FHIT in Human Cancer

telomere Exons 10 9

145

a

8

~1

n 6 m n

2 3 BA

z

8

7

6

IHPVl61

n 5

1 t3;8 1 I

3

4

centrornere

n 2

1

Y648D4 Y850A6 YAC75OF1

Fig. 1 Organization of the FHITgene. Approximate location of the YAC clones, the relevant STSs, plasmid and HPV16 integration sites, and the t(3;8) translocation breakpoint are indicated.

experiments aimed at identifying genes within the deleted region. An oligonucleotide primer designed from the initial trapped exon was used in primer extension to obtain a 5’ extended product of the cDNA by a rapid amplification of cDNA ends (RACE) reaction. The longest product from the RACE reaction detected a ubiquitously expressed 1.1-kb mRNA by Northern blot analysis of mRNA from various normal tissues. Because cDNA sequences 5’ and 3‘ of the first trapped exon (exon 5 in Fig. 1)were not within the cosmid contig, cosmid libraries from YACs 850A6 and 750F1, which extend centromeric and telomeric to the fragile region deletions, respectively, were screened with the 5’ and 3’ cDNA probes flanking exon 5. Cosmids containing the remaining exons were then used to derive intron sequences using cDNA primers, and the structure of the gene was determined as shown in Fig. 1. The 1.1-kb FHITcDNA consists of 10 small exons and is distributed over a genomic locus of about a megabase. Only exon 5 falls within the region of homozygous deletion originally observed in tumor-derived cell lines. The coding region of the open reading frame begins in exon 5 and ends in exon 9. Interestingly, the first three exons (El, E2, and E3 of Fig. 1)of the gene are centromeric to the t(3;8) break. Thus this gene has become a strong candidate for involvement in initiation of the clear cell renal carcinoma of the t(3;8) family because one copy of the gene is disrupted by the translocation. In addition, the location of several markers, such as BE758-6 (Lisitsyn and Wigler, 1995) and D3S1300, previously found deleted at high frequency in tumors and cell lines (Kastury et al., 1996), within the FHIT gene locus, close to the first coding exon 5, suggested that FHITis the target of these deletions. Analysis of FHIT expression in normal and tumor tissues by Northern blot revealed a low level of expression in all normal human tissues tested, whereas varying levels of FHIT transcripts, from barely detectable to almost normal levels, were found in tumor-derived cell lines (Ohta et al., 1996). Reverse transcription-polymerase chain reaction (PCR) was used to

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detect abnormalities in FHZT transcripts in primary gastrointestinal tumors; a pattern of products ranging from one apparently normal-sized amplified transcript to numerous aberrant bands without a normal sized band was observed. Sequence analysis of the usually shorter aberrant products revealed an absence of various regions between exons 4 and 9, whereas normal tissue mRNA from the same organ did not exhibit alteration in the amplified sequences (Ohta et al., 1996). Because the aberrant transcripts frequently lacked exon 5 , which begins the open reading frame of FHIT, or exon 8, the highly conserved histidine triad-containing domain, it is likely that these aberrant products could not encode functional proteins. Insertions of various lengths of DNA, either between or replacing exons, were also observed (Ohta et al., 1996).

111. THE Fhit PROTEIN AND ITS BIOCHEMICAL PROPERTIES The FHlTgene encodes a polypeptide of 16.8 kD that is composed of 147 amino acids that show 52% identity and 69% similarity to a core region of 109 amino acids of the diadenosine S’,S’’’-Pl,P4-tetraphosphate (Ap,A) hydrolase from the fission yeast Schizosaccaromyces pombe (Robinson et al., 1993; Huang et al., 1995; Ohta et al., 1996). The latter enzyme is a 182 amino acid protein that catalyzes the in vitvo hydrolysis of dinucleoside polyphosphates, with Ap,A as the preferred substrate. Both the Fhit protein and the S. pombe Ap,A hydrolase are related by sequence to the HIT proteins, a group of molecules of unknown functions characterized by four conserved histidines, three of which make up a histidine triad (HIT) sequence, H X H X H, where X is most frequently valine (Huang et al., 1995; Ohta et al., 1996). Subsequent biochemical studies (Barnes et al., 1996) clearly demonstrated that the human Fhit protein could be classified as an Ap,A hydrolase on the basis of its in uitro enzymatic activity. Ap,A is the preferred substrate among ApnAs, and AMP is always one of the reaction products. By site-directed mutagenesis, each of the four conserved histidine residues of FHZT was changed to an asparagine. Each change resulted in a decrease in Ap,A hydrolase activity, demonstrating that all four conserved histidines are required for full activity, but the central histidine of the triad (H96) is absolutely essential for Ap,A hydrolase activity (Barnes et al., 1996). Consequently, alterations of exon 8 could be critical for the biological activity of the Fhit protein. The crystal structure of histidine triad nucleotide-binding protein (HINT) showed that histidine triad proteins (HIT) are nucleotide-binding proteins (Brenner et al., 1997). HINT-nucleotide complexes demonstrated that the

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most conserved residues in the superfamily mediate nucleotide binding and that the HIT motif forms part of the phosphate binding loop. Thus, FHIT is an enzyme of the HIT family, a family of genes involved in nucleotide metabolism. FHIT substrates Ap,A and Ap,A have been considered alarmones in bacterial systems, which are produced in times of cellular stress (Kitzler et al., 1992). Accumulation of Ap,A in human cultured cells is induced by interferon (Vartanian et al., 1996), and Ap,A is reported as a consequence of contact inhibition of growth and toxic stress (Segal and Le Pecq, 1986). FHIT-substrate or substrate-enzyme complexes may thus be involved in signaling responses to cellular stress, resulting in cell cycle arrest.

IV. FHIT ABNORMALITIES IN HUMAN CANCER For a summary of FHIT abnormalities, see Table I.

A. Tumors of the Gastrointestinal Tract The initial finding of a high incidence (46%) of LOH at 3p14.2 in gastric cancer and the definition of a small region of homozygous deletion (60 kb) in a gastric carcinoma cell line (KatoIII) (Kastury et al., 1996) contributed to the positional cloning of the 1-Mb FHIT gene, which includes FRA3B (Ohta et al., 1996). In the latter study, abnormal expression of FHIT was reported in 5 of 10 esophageal cancers, in 5 of 9 stomach cancers, and in 3 of 8 colon cancers. Subsequently, three other studies investigated FHIT abnormalities in colon and gastric tumors and cell lines (Thiagalingam et al., 1996; Baffa, submitted for publication; Gemma et al., 1997). Because most of the initial results on the FHIT gene were obtained mainly by nested RT-PCR analysis of gene expression, some investigators cautioned that such analysis may produce artifactual aberrant products not directly related to an alteration of the FHIT gene and its function. In fact, Thiagalingam et al. (1996) reported LOH at loci internal to the FHIT gene in only 22% of the colon cancer xenografts examined, and abnormal transcription of the gene was found in a minority ( 4 of 31) of the same tumors. They suggested that FHIT may not be causally related to colon cancer pathogenesis or that it is a bystander casualty of the genomic instability of this fragile site. The development of specific antibodies recognizing the Fhit protein has allowed a correlation of DNA lesions at the FHIT locus with aberrant RT-PCR products and altered Fhit protein in cell lines derived from tumors of the gastrointestinal tract, as well as from other sites (Druck et al., 1997). In this study the authors demonstrated that even when DNA and RNA alterations

Table I Summary of FHIT Abnormalities in Tumors Tumor type

Gastrointestinal Esophageal Colon

Gastric

Aerodigestive Lung

Type of specimena

10T 31 T 8T 9T 32 T 6 CL 40 T

14 SCLC (T) 45 NSCLC (T) 17 SCLC (CL) 24 NSCLC (CL) 1 s SCLC (CL)

FHIT abnormalitiesb

Types of Assay'

Reference

5/10 (50) 7/31 (22) 4/31 (13) 3/8 (37) 5/9 18/32 (56) 4/8 (50) 16/38 (42) 1/40

RT-PCR LOH RT-PCR RT-PCR RT-PCR LOH, SB, RT-PCR SB, RT-PCR, WB LOH SSCP

Otha et al. (1996) Thiagahgametal. (1996) Thmgahgam d al. (1996) Otha et al. (1996) Otha et al. (1996) Baffa et al. (1997) Baffa et al. (1997) Gemma et al. (1997) Gemma et al. (1997)

11/14 (80) 18/45 (40) 63-92% 0117 ( 0 ) 7/24 (29) 100% LOH; 73% RT-PCR

RT-PCR RT-PCR LOH DNAPCR, SB, RT-PCR DNA-PCR, SB, RT-PCR

Sozzi et al. (1996) Sozzi et al. (1996) Yanagisawa et al. (1996) Yanagisawa et al. ( 1996) Fong et a/. (1997)

Head and neck

17 NSCLC (CL) 8 SCLC (T) 108 NSCLC (T) 8 CIS 26 CL

16 CL Breast

Merkel cell carcinoma Pleomocphic adenoma parotid gland

11 Cl 30 T 3 14 T 1T

88% LOH; 77% RT-PCR 100% LOH 45% LOH; 59% RT-PCR 6/8 (75) 15/25 (55) 13/20 (65) 3/22 (12) 13/16 (81) 7/16 (45) 3/11 (27) 9/30 (30) 3/3 (100) 8/14 (57) 1/1

LOH RT-PCR FISH SB LOH RT-PCR RT-PCR RT-PCR DNA-PCR, RT-PCR RT-PCR Cytogenetics, FISH sequence, RT-PCR

Fong et al. ( 1 997) Fong et al. (1997) Fong et al. (1997) Fong et a[. (1997) Virgilio et al. (1996)

Mao et al. ( 1996) Negrini etal. (1996) Negrini et al. (1996) Panagopodoset al. (1996) Sozzi et al. (1996) Geurts et al. (1997)

dT, tumors; CL, cell line; AH, atypical hyperplasia; CIS, carcinoma in situ. *Number of cases. Numbers in parentheses represent percentage of total number of cases.